Ferroptosis and FASN

Ferroptosis, a form of programmed cell death characterized by iron-dependent lipid peroxidation, is a promising avenue in cancer therapy. New research shows that disrupting cancer cells' lipid metabolism can enhance their susceptibility to ferroptosis. Orlistat, an FDA-approved anti-obesity drug known for inhibiting fatty acid synthase (FASN), plays a pivotal role in this context.

A study by Lian and colleagues demonstrated that restricting cancer cells' access to fats increases their sensitivity to ferroptosis. By inhibiting FASN, Orlistat effectively reduces lipid synthesis within cancer cells, thereby promoting ferroptosis. This mechanism was observed in lung cancer cells, where orlistat inhibited cell proliferation and induced ferroptosis-like cell death. 

Combining orlistat with ferroptosis inducers could be an effective strategy for cancer treatment. By simultaneously disrupting lipid metabolism and inducing ferroptosis, this approach could potentially overcome resistance mechanisms that cancer cells employ against conventional therapies.

References

Sokol, K. H., et al (2024) Lipid availability influences ferroptosis sensitivity in cancer cells by regulating polyunsaturated fatty acid trafficking. Cell Chemical Biology. doi.org/10.1016/j.chembiol.2024.09.008.

Zhou, W., Zhang, J., Yan, M. et al. Orlistat induces ferroptosis-like cell death of lung cancer cells. Front. Med. 15, 922–932 (2021). https://doi.org/10.1007/s11684-020-0804-7

Enhanced Cancer Immunotherapy by Targeting Monoamine Oxidase A (MAO-A)

Monoamine oxidase A (MAO-A), an enzyme historically studied for its role in neurotransmitter metabolism, has emerged as a promising target in cancer immunotherapy. By inhibiting MAO-A, researchers have found a potential to improve immune responses within the tumor microenvironment, particularly by enhancing the function of immune cells such as CD8+ T cells and tumor-associated macrophages (TAMs). Findings from recent studies demonstrate that MAO-A inhibition can significantly reduce tumor growth, presenting a dual-purpose pathway that leverages known MAO-A inhibitors for both neurological and oncological applications.

MAO-A metabolizes monoamines like serotonin, dopamine, and norepinephrine in the brain, influencing emotional and behavioral states. However, recent studies reveal that MAO-A regulates immune cell functions, especially within the tumor microenvironment. This dual role makes MAO-A a unique target in cancer, as inhibiting it may enhance the body’s antitumor immune response. For instance, high MAO-A activity has been linked to immune suppression, fostering a pro-tumor environment by regulating immune cell metabolism and suppressing T-cell activity.

The reviewed study highlights how MAO-A inhibition upregulates key antitumor cytokines, such as interferon-gamma (IFN-γ), and cytotoxic molecules like granzyme B. These findings support the theory that reducing MAO-A activity alleviates immune cell exhaustion and promotes the proliferation and effectiveness of tumor-infiltrating T-cells, essential players in targeting cancer cells.

One of the critical mechanisms by which MAO-A inhibition supports antitumor immunity is through serotonin, a neurotransmitter often degraded by MAO-A. CD8+ T cells produce serotonin as an activation signal, which supports their proliferation and cytotoxic function. However, high MAO-A activity in the tumor environment degrades serotonin, leading to suppressed T-cell activity. Inhibiting MAO-A has been shown to maintain serotonin levels, facilitating a robust T cell response and enhancing T cell activation through serotonin signaling pathways.

According to recent findings, MAO-A inhibitors like phenelzine, clorgyline, and moclobemide significantly enhance serotonin-mediated T-cell activation, amplifying downstream pathways critical for immune responses. These inhibitors effectively prevent immune cell exhaustion by maintaining elevated serotonin levels, promoting a sustained immune assault on tumor cells.

MAO-A inhibitors could become valuable alongside existing immune checkpoint inhibitors (ICIs), such as PD-1/PD-L1 blockers. MAO-A inhibition reduces the expression of exhaustion markers in T cells, allowing these cells to better respond to checkpoint inhibition therapies. Combining MAO-A inhibitors with anti-PD-1 therapy demonstrated enhanced antitumor efficacy, with significant tumor suppression in preclinical models.

Furthermore, MAO-A inhibition has been shown to impact other immunosuppressive cells in the tumor microenvironment, such as TAMs. MAO-A influences TAM polarization by promoting an immunosuppressive phenotype through increased reactive oxygen species (ROS) production, which fosters oxidative stress and dampens immune responses. Blocking MAO-A reduces ROS levels, reprogramming TAMs toward a pro-inflammatory state, thereby boosting the immune response against the tumor.

Given MAO-A inhibitors’ long history in treating neurological disorders, side effects related to serotonin syndrome and hypertensive crises from dietary tyramine intake are known challenges. The reviewed research suggests combining MAO-A inhibitors with nanoformulations, such as cross-linked multilamellar liposomal vesicles, could mitigate these side effects while preserving antitumor efficacy. This approach has already demonstrated superior results in preclinical melanoma models, offering a promising pathway for safer clinical applications.

Methylene Blue (MB) may be a safe alternative to traditional MAO-A inhibitors for enhancing antitumor immunity. Unlike irreversible MAO-A inhibitors such as phenelzine and clorgyline, which carry risks of serotonin syndrome and hypertensive crises when combined with certain foods or medications, MB acts as a reversible MAO-A inhibitor. This reversible action maintains MAO-A activity at a modulated level, reducing the likelihood of adverse interactions, but also leverages MB’s inherent antioxidant properties, which help regulate reactive oxygen species (ROS) levels in immune cells. By balancing oxidative stress and preserving serotonin within the tumor microenvironment, MB may sustain CD8+ T cell activity and support TAM reprogramming without the side effects associated with traditional MAO-A inhibitors. This dual action could make MB a compelling candidate for cancer immunotherapy, presenting a safe, accessible approach to amplifying immune responses against tumors.

The dual role of MAO-A in both neurological and immunological regulation makes it an exciting target in cancer therapy. Repurposing MAO-A inhibitors offers a feasible approach to enhance antitumor immunity, leveraging their established safety profiles for new therapeutic avenues. 

References

Wang, Xi & Li, Bo & Kim, Yu & Wang, Yu-Chen & Li, Zhe & Yu, Jiaji & Zeng, Samuel & Ma, Xiaoya & Choi, In Young & Di Biase, Stefano & Smith, Drake & Zhou, Yang & Li, Yan-Ruide & Ma, Feiyang & Huang, Jie & Clarke, Nicole & To, Angela & Gong, Laura & Pham, Alexander & Yang, Lili. (2021). Targeting monoamine oxidase A for T cell–based cancer immunotherapy. Science Immunology. 6. eabh2383. 10.1126/sciimmunol.abh2383. 

Ma, Y., Chen, H., Li, H. et al. Targeting monoamine oxidase A: a strategy for inhibiting tumor growth with both immune checkpoint inhibitors and immune modulators. Cancer Immunol Immunother 73, 48 (2024). https://doi.org/10.1007/s00262-023-03622-0

Gillman, Ken & Ng, Bradley & Cameron, Andrew & Liang, Rhea. (2008). Methylene blue is a potent monoamine oxidase inhibitor. Canadian Journal of Anesthesia/Journal canadien d'anesthésie. 55. 311-312. 10.1007/BF03017212. 

A pan-cancer screen identifies drug combination benefit in cancer cell lines

This study, a collaboration between researchers from the Netherlands Cancer Institute, Wellcome Sanger Institute (UK), Delft University of Technology, and Oncode Institute, outlines a comprehensive pan-cancer screening to identify effective drug combinations across diverse cancer cell lines. Screening 51 drug combinations in 757 cell lines, the team classified responses into synergy, Bliss additivity, and independent drug action (IDA), recognizing that effective responses extend beyond traditional synergy.

A key concept introduced is efficacious combination benefit (ECB), which identifies combinations that achieve over 50% cell viability reduction, regardless of interaction type. ECB proved more predictive of response in patient-derived xenografts (PDX) than synergy alone, offering a more robust framework for preclinical evaluation.

Top-performing combinations include:

AZD7762 + Gemcitabine: Effective with high synergy rates in the large intestine, esophagus, and endometrium cells.

Navitoclax + Axitinib: Showed strong synergy in bladder cancer cell lines.

MK-1775 + Cisplatin: Displayed high Bliss efficacy across soft tissue, thyroid, kidney, and nervous system cancers.

Trametinib + LGK974: Demonstrated robust IDA across many tissue types.

Olaparib + Temozolomide: Increased efficacy in bone cancers when stratified by biomarkers.

This collaborative work provides a valuable preclinical framework for exploring drug combinations with high ECB scores across cancer types, guiding future clinical applications based on biomarker-driven selection.

Reference

Vis DJ, Jaaks P, Aben N, Coker EA, Barthorpe S, Beck A, Hall C, Hall J, Lightfoot H, Lleshi E, Mironenko T, Richardson L, Tolley C, Garnett MJ, Wessels LFA. A pan-cancer screen identifies drug combination benefit in cancer cell lines at the individual and population level. Cell Rep Med. 2024 Aug 20;5(8):101687. doi: 10.1016/j.xcrm.2024.101687. PMID: 39168097; PMCID: PMC11384948.

Repurposing Orlistat: Anti-Cancer Mechanisms and Potential in Therapeutics

Orlistat, developed initially and FDA-approved as an anti-obesity drug, inhibits pancreatic and gastric lipases, thereby reducing dietary fat absorption. However, emerging research over the past two decades has positioned Orlistat as a potent anti-cancer agent. Its anti-tumor effects largely stem from its role as an inhibitor of fatty acid synthase (FASN), an enzyme essential for lipid metabolism that is often upregulated in cancer cells to support rapid proliferation, survival, and metastasis. Cancer cells rely heavily on de novo lipogenesis, driven by FASN, to meet their high energy demands and facilitate membrane synthesis. By targeting this pathway, Orlistat disrupts lipid metabolism and induces cytotoxic effects in various cancer models.

Mechanisms of Action in Cancer Therapy

Orlistat’s anti-cancer effects are multi-faceted, involving lipid metabolism disruption, apoptosis induction, ferroptosis-like cell death, and antiangiogenic activity. Below is a breakdown of these mechanisms based on the recent studies:

1. FASN Inhibition and Apoptosis Induction

Orlistat’s primary anti-cancer mechanism involves the inhibition of FASN. By blocking FASN, Orlistat effectively cuts off the cancer cell’s supply of fatty acids essential for cell membrane synthesis and energy production. This disruption forces cancer cells into metabolic stress, leading to cell cycle arrest and apoptosis. Studies across multiple cancer types, including breast, prostate, lung, and pancreatic cancers, have demonstrated that Orlistat can significantly reduce tumor cell proliferation and induce apoptosis by modulating the expression of apoptotic proteins such as Bax, Bcl-2, and caspase-3.

Orlistat induces significant cytotoxicity in breast cancer cells and enhances apoptotic markers without affecting normal breast cells, highlighting its selective toxicity. This selectivity positions Orlistat as a potentially safe and effective treatment option. Similarly, in pancreatic cancer, Orlistat reduces proliferation and promotes apoptosis by targeting FASN, interrupting the metabolic pathways essential for tumor growth.

2. Ferroptosis and Ferroptosis-Like Cell Death

A novel discovery regarding Orlistat’s mechanism is its induction of ferroptosis-like cell death, particularly noted in lung cancer cells. Ferroptosis is an iron-dependent form of cell death characterized by lipid peroxidation. Orlistat achieves this by downregulating GPX4, a critical enzyme that protects against lipid peroxidation, and increasing reactive oxygen species (ROS) levels. The accumulation of ROS leads to oxidative damage and, ultimately, cell death. This ferroptotic pathway adds a layer of lethality to Orlistat’s anti-tumor effects, making it particularly effective against cancers with high oxidative stress responses.

3. Antiangiogenic Effects

Orlistat has shown promising antiangiogenic properties by inhibiting endothelial cell proliferation, an essential step in tumor angiogenesis. Inhibition of FASN in endothelial cells by Orlistat blocks fatty acid synthesis, which is critical for new blood vessel formation. Additionally, Orlistat downregulates the expression of VEGFR2, a receptor essential for vascular endothelial growth factor (VEGF) signaling in angiogenesis. This antiangiogenic effect disrupts the tumor’s ability to establish a blood supply, thereby limiting its growth and metastatic potential. The antiangiogenic effects have been observed in breast and colorectal cancer models, emphasizing Orlistat’s potential in targeting vascular-dependent tumors.

4. Anti-Inflammatory Effects and Immune Modulation

In inflammation-driven cancers, such as colitis-associated colon cancer, Orlistat reduces tumor-promoting inflammation by inhibiting the NF-κB and STAT3 pathways. These pathways regulate pro-inflammatory cytokine production and cell survival in the tumor microenvironment. By blocking these pathways, Orlistat decreases chronic inflammation, reducing the risk of progression from chronic inflammatory conditions to cancer. This is particularly relevant in cancers influenced by the Western diet, where chronic inflammation contributes to tumor initiation and growth.

Orlistat’s immune-modulatory effects extend to its role in promoting the differentiation and activation of macrophages, critical players in the anti-tumor immune response. In T-cell lymphoma models, Orlistat enhances myelopoiesis and promotes the differentiation of bone marrow cells into M1 macrophages, a tumoricidal phenotype. This boosts the body’s natural immune response against tumors, making Orlistat a promising agent for immunomodulatory therapy.

5. Metabolic Reprogramming and Combination with Metabolic Inhibitors

Cancer cells often adapt to metabolic stress by shifting between glycolysis and lipid metabolism. In gastric cancer models, Orlistat has shown increased efficacy when combined with PGM1 knockdown, which limits glycolytic activity. Under glucose-deprived conditions, the knockdown of PGM1 alongside Orlistat treatment prevents cancer cells from compensating with lipid metabolism, enhancing Orlistat’s anti-cancer effects. This combination targets cancer cells' metabolic flexibility, exploiting their dependency on glucose and fatty acid pathways to sustain growth.

6. Delay in Hepatocarcinogenesis

Studies have shown that Orlistat delays the onset and progression of hepatocellular carcinoma (HCC) in animal models with hepatic co-activation of AKT and c-Met, common pathways in liver cancer. Orlistat impairs AKT/SREBP1/FASN signaling, reducing lipogenesis and cell proliferation. This mechanism suggests that Orlistat could be beneficial in managing HCC, especially in patients with metabolic dysfunctions that heighten their risk for liver cancer.

Mechanisms of Orlistat’s Anti-Cancer Activity

Orlistat’s multi-targeted approach offers a promising route for treating various types of cancer, particularly those reliant on lipid metabolism. Its ability to induce selective cytotoxicity, modulate immune responses, and disrupt cancer metabolism highlights its versatility as an anti-cancer agent. However, challenges remain, primarily related to its bioavailability and off-target effects.

Studies have demonstrated Orlistat’s efficacy in in vitro and in vivo models, but its low systemic bioavailability when administered orally limits its effectiveness in specific cancers. 

Another consideration is patient selection. Orlistat may be particularly effective for patients with cancers characterized by metabolic dysregulation, such as those with obesity-driven cancers or metabolic syndrome. Further studies are needed to identify specific biomarkers that can predict response to Orlistat and guide its clinical use.

Orlistat, though developed initially for obesity management, holds substantial promise in oncology due to its multi-faceted mechanisms that target cancer cell metabolism, induce cell death, and modulate immune responses. Its efficacy across various cancers, including breast, lung, pancreatic, colon, and liver, underscores its potential as a repurposed therapeutic. 

References

Kant, Shiva & Kumar, Ajay & Singh, Sukh. (2013). Myelopoietic Efficacy of Orlistat in Murine Hosts Bearing T Cell Lymphoma: Implication in Macrophage Differentiation and Activation. PloS one. 8. e82396. 10.1371/journal.pone.0082396. 

Kant, Shiva & Kumar, Ajay & Singh, Sukh. (2012). Fatty acid synthase inhibitor orlistat induces apoptosis in T cell lymphoma: Role of cell survival regulatory molecules. Biochimica et biophysica acta. 1820. 1764-73. 10.1016/j.bbagen.2012.07.010. 

Schcolnik-Cabrera, Alejandro & Chavez, Alma & Gomez, Guadalupe & Taja, Lucia & Cardenas-Barcenas, Rocio & Trejo-Becerril, Catalina & Perez-Cardenas, Enrique & Gonzalez-Fierro, Aurora & Duenas-Gonzalez, Alfonso. (2018). Orlistat as a FASN inhibitor and multitargeted agent for cancer therapy. Expert Opinion on Investigational Drugs. 27. 10.1080/13543784.2018.1471132. 

Dowling, Shawn & Cox, James & Cenedella, Richard. (2009). Inhibition of Fatty Acid Synthase by Orlistat Accelerates Gastric Tumor Cell Apoptosis in Culture and Increases Survival Rates in Gastric Tumor Bearing Mice In Vivo. Lipids. 44. 489-98. 10.1007/s11745-009-3298-2. 

Sokolowska, Ewa & Presler, Malgorzata & Goyke, Elzbieta & Milczarek, Ryszard & Swierczynski, Julian & Sledzinski, Tomasz. (2017). Orlistat Reduces Proliferation and Enhances Apoptosis in Human Pancreatic Cancer Cells (PANC-1). Anticancer research. 37. 6321-6327. 10.21873/anticanres.12083. 

Jovankić, Jovana & Nikodijević, Danijela & Milutinović, Milena & Nikezic, Aleksandra & Kojić, Vesna & Cvetković, Aleksandar & Cvetkovic, Danijela. (2022). Potential of Orlistat to induce apoptotic and antiangiogenic effects as well as inhibition of fatty acid synthesis in breast cancer cells. European Journal of Pharmacology. 939. 175456. 10.1016/j.ejphar.2022.175456. 

Elmasry, Thanaa & El-Nagar, Maysa & Oriquat, Ghaleb & Alotaibi, Badriyah & Saad, Hebatallah M. & El Zahby, Enas & Ibrahim, Hanaa. (2024). Therapeutic efficiency of Tamoxifen/Orlistat nanocrystals against solid ehrlich carcinoma via targeting TXNIP/HIF1-α/MMP-9/P27 and BAX/ Bcl2/P53 signaling pathways. Biomedicine & Pharmacotherapy. 180. 10.1016/j.biopha.2024.117429. 

Zhang, Cong & Sheng, Lei & Yuan, Ming & Hu, Junjie & Meng, Yan & Wu, Yong & Chen, Liang & Yu, Huifan & Li, Shan & Zheng, Guohua & Qiu, Zhenpeng. (2020). Orlistat delays hepatocarcinogenesis in mice with hepatic co-activation of AKT and c-Met. Toxicology and Applied Pharmacology. 392. 114918. 10.1016/j.taap.2020.114918. 

Xu, Guangxu & Zhao, Ziyi & Wysham, Weiya & Roque, Dario & Fang, Ziwei & Sun, Wenchuan & Yin, Yajie & Deng, Boer & Shen, Xiaochang & Bae-Jump, Victoria. (2023). Orlistat exerts anti-obesity and anti-tumorigenic effects in a transgenic mouse model of endometrial cancer. Frontiers in Oncology. 13. 10.3389/fonc.2023.1219923. 

Cervantes Madrid, Diana & Gomez, Guadalupe & Gonzalez‑Fierro, Aurora & Perez-Cardenas, Enrique & Taja, Lucia & Trejo-Becerril, Catalina & Duenas-Gonzalez, Alfonso. (2017). Feasibility and antitumor efficacy in vivo, of simultaneously targeting glycolysis, glutaminolysis and fatty acid synthesis using lonidamine, 6-diazo-5-oxo-L-norleucine and orlistat in colon cancer. Oncology Letters. 13. 10.3892/ol.2017.5615. 

Zhou, Wenjing & Zhang, Jing & Yan, Mingkun & Wu, Jin & Lian, Shuo & Sun, Kang & Li, Baiqing & Ma, Jia & Xia, Jun & Lian, Chaoqun. (2021). Orlistat induces ferroptosis-like cell death of lung cancer cells. Frontiers of Medicine. 15. 10.1007/s11684-020-0804-7. 

Ye, Mujie & Lu, Feiyu & Chen, Jinhao & Yu, Ping & Xu, Yanling & He, Na & Hu, Chunhua & Zhong, Yuan & Yan, Lijun & Gu, Danyang & Xu, Lin & Bai, Jianan & Tian, Ye & Tang, Qiyun. (2023). Orlistat Induces Ferroptosis in Pancreatic Neuroendocrine Tumors by Inactivating the MAPK Pathway. Journal of Cancer. 14. 1458-1469. 10.7150/jca.83118. 

Mojjarad, Behnam & Pazhang, Yaghub. (2020). Orlistatand and Rosuvastatin Suppressed Proliferation of K562 Human Myelogenous Leukemia Cell Line through AMPK/Akt/c-Myc Signaling. 10.21203/rs.3.rs-18746/v2. 

Discovery of Stem-Like CD4 T Cells

Researchers at Emory University's Winship Cancer Institute have uncovered a previously unknown immune cell type, a stem-like CD4 T cell, which shows promise to improve cancer immunotherapy. Led by Dr. Haydn T. Kissick and published in Nature, this study highlights the transformative potential of these cells in enhancing anti-tumor immunity. The breakthrough is relevant to patients whose cancers resist existing immunotherapies, potentially marking a shift toward broader therapeutic success.

This newly discovered CD4 T cell population resides primarily in lymph nodes adjacent to tumors, maintaining a state of dormancy that limits their immune activity. Although they can drive a potent anti-tumor response, these cells are frequently inactive, allowing tumors to evade immune attacks. According to Dr. Kissick, in cases where these cells are active, around 10% of patients, a robust immune response against cancer emerges, leading to extended survival and heightened responsiveness to immune checkpoint inhibitors, specifically PD1 blockade therapy.

The study’s findings emphasize the regenerative capabilities of these stem-like CD4 T cells, which can renew themselves and transform into various immune cell types. This regenerative potential is regulated by two specific markers, PD1 and TCF1, which modulate their self-renewal and differentiation pathways. When activated in lab models, these cells enhance the efficacy of PD1 blockade therapy, a prominent treatment modality in cancer immunotherapy.

The study’s first author, Dr. Maria Cardenas, highlights the importance of reactivating these cells from their suppressed, “idle” state to a more active anti-tumor role. This suppression often acts as a blockade that limits the immune system’s response to cancer, making it critical to identify mechanisms to switch these cells to an active state. Dr. Kissick says that most cancer patients possess stem-like CD4 T cells, and understanding how to shift their states could revolutionize the field of cancer treatment, opening up avenues to enhance immunotherapy responses across a broader patient spectrum.

A promising path forward involves exploring mRNA and lipid nanoparticle (LNP) technology to reprogram these stem-like CD4 T cells, effectively removing the “brakes” on the immune system's response to cancer. Researchers use mRNA technology to manipulate the cells' behavior, encouraging a continuous and sustained anti-tumor response. This approach could potentially lead to a novel class of adaptable and highly targeted immunotherapies, making them effective against cancers that were previously difficult to treat with existing therapies.

The identification of stem-like CD4 T cells introduces a new approach to cancer immunotherapy. By activating these dormant but powerful immune cells, this approach has the potential to enhance treatment efficacy. 

For more details, refer to the original study by Cardenas et al.

Repurposing metformin for cancer treatment

Across various cancer types and contexts, metformin has demonstrated promising anti-tumor mechanisms that include modulating cellular energy metabolism, enhancing immune responses, inhibiting metastatic processes, and inducing cell death pathways. Below is a structured synthesis of the main mechanisms through which metformin exerts its effects on cancer cells, alongside a simple table summarizing these key mechanisms.

Metformin's Modulation of Immune Response and PD-L1 Regulation 

Metformin has shown efficacy in enhancing the therapeutic effects of anti-PD-L1 antibodies through its impact on the gut microbiota. Studies in mouse models have demonstrated that metformin's regulation of gut microbiota boosts anti-tumor immunity, increasing CD8+ T-cell infiltration and IFN-γ expression, crucial components of the immune response against tumors. Additionally, a metformin-liposome combination reduced PD-L1 expression. It reversed hypoxia in tumor tissues, which amplified the immune reaction by alleviating T-cell exhaustion in photodynamic therapy (PDT) contexts.

Inhibition of Nrf2 Signaling and Reduction of Chemoresistance

Metformin's effect on Nrf2 signaling has gained attention for its role in reducing cancer chemoresistance. It downregulates Nrf2 transcriptional activity, often associated with resistance in cancers like hepatocellular carcinoma (HCC), and may be linked to improved sensitivity to chemotherapeutic drugs, such as cisplatin. This mechanism is vital in overcoming drug resistance that characterizes aggressive cancers.

Induction of Ferroptosis via Inhibition of UFMylation of SLC7A11

Metformin induces ferroptosis, a form of regulated cell death, in breast cancer cells by inhibiting the UFMylation process of SLC7A11. This pathway facilitates the accumulation of iron-dependent lipid peroxidation, lethal to cancer cells. Metformin and other ferroptosis-inducing agents have synergistic effects in enhancing cell death in specific breast cancer models, highlighting ferroptosis as a novel anti-cancer pathway mediated by metformin.

Inhibition of the Epithelial-Mesenchymal Transition (EMT) and Metastasis

Metformin has been observed to inhibit the EMT, a critical process in cancer metastasis, particularly in pancreatic cancer cells. By downregulating TGF-β1-induced EMT signaling, metformin reduces cancer cells' migration potential, limiting their ability to metastasize to distant organs. This pathway, involving the inhibition of Smad and Akt/mTOR pathways, demonstrates metformin's potential in reducing metastasis, a significant factor in cancer progression and patient prognosis.

Targeting Hypoxic Tumor Microenvironments by Modulating PDH and HIF-1α

Under hypoxic conditions commonly present in solid tumors, metformin increases pyruvate dehydrogenase (PDH) levels. It suppresses HIF-1α, a critical factor promoting survival in low-oxygen conditions. This reduces cancer cell proliferation and migration and enhances apoptosis, particularly in oral squamous cell carcinoma models. Metformin disrupts cancer cells’ metabolic adaptations that support growth and survival in oxygen-poor environments by targeting hypoxia-driven pathways.

Metformin’s Effect on Glutamine Metabolism and Autophagy

In various cancer cell types, metformin reduces glutaminase activity, leading to lower levels of glutamate and ammonia. This reduction limits autophagy, a survival mechanism in tumors under metabolic stress. By impairing glutamine metabolism, metformin disrupts cellular homeostasis, causing cell death, particularly in cancer cells reliant on high glutaminase activity for growth.

Influence on Cancer Stem Cells

Metformin reduces cancer stem cell (CSC) populations in colorectal cancer by altering glutamine metabolism, which CSCs rely on. This mechanism weakens CSCs' self-renewal capabilities, rendering them more susceptible to conventional therapies and reducing the likelihood of relapse.

Targeting Hypoxia-Driven HIF-1 Pathway in Multiple Myeloma

Hypoxia-inducible factor-1α (HIF-1α) significantly promotes tumor survival in low-oxygen conditions by enhancing glycolysis and angiogenesis. Metformin inhibits HIF-1α, suppressing these adaptive responses and leading to apoptosis in hypoxic cells in multiple myeloma and other cancers. This makes metformin a promising adjuvant to therapies targeting hypoxia-adapted cancer cells.

Inhibition of TGF-β-Induced EMT and Fibrosis

Metformin’s inhibition of TGF-β-induced EMT extends beyond pancreatic cancer, affecting other cancers and even fibrotic diseases. EMT is essential for metastasis, and metformin’s suppression of this process, notably through the downregulation of TGF-β/Smad signaling, limits the ability of cancer cells to invade new tissues.

Enhanced PDH Activity and Reduced HIF-1α in Hypoxic Conditions

Under hypoxia, metformin promotes pyruvate dehydrogenase (PDH) activity and reduces HIF-1α levels, impairing the Warburg effect and promoting apoptosis in cancers like oral squamous cell carcinoma. This dual action on energy metabolism and hypoxia adaptation is vital in metformin’s anti-tumor arsenal​.

Table: Metformin's Main Anti-Cancer Mechanisms

Please refer to the spreadsheet for more details on Metformin's synergistic combinations and effects against over 30 anticancer variables.

References

Guimarães, Talita & Farias, Lucyana & Fraga, Carlos Alberto & Paula, Alfredo & Santos, Sérgio & Gomez, Ricardo & Guimaraes, Andre. (2017). Abstract 5424: Metformin increases PDH and suppresses HIF1A under hypoxic conditions and induces cell death in oral squamous cell carcinoma. Cancer Research. 77. 5424-5424. 10.1158/1538-7445.AM2017-5424. 

Yoshida, Juichiro & Ishikawa, Takeshi & Endo, Yuki & Matsumura, Shinya & Ota, Takayuki & Mizushima, Katsura & Hirai, Yasuko & Oka, Kaname & Okayama, Tetsuya & Sakamoto, Naoyuki & Inoue, Ken & Kamada, Kazuhiro & Uchiyama, Kazuhiko & Takagi, Tomohisa & Naito, Yuji & Itoh, Yoshito. (2020). Metformin inhibits TGF‑β1‑induced epithelial‑mesenchymal transition and liver metastasis of pancreatic cancer cells. Oncology Reports. 44. 10.3892/or.2020.7595. 

Deng, Xin-Sheng & Wang, Shuiliang & Deng, Anlong & Liu, Bolin & Edgerton, Susan & Lind, Stuart & Wahdan-Alaswad, Reema & Thor, Ann. (2012). Metformin targets Stat3 to inhibit cell growth and induce apoptosis in triple-negative breast cancers. Cell cycle (Georgetown, Tex.). 11. 367-76. 10.4161/cc.11.2.18813. 

Yang, Jingjing & Zhou, Yulu & Xie, Shuduo & Wang, Ji & Li, Zhaoqing & Chen, Lini & Mao, Misha & Chen, Cong & Huang, Aihua & Chen, Yongxia & Zhang, Xun & Khan, Noor Ul Hassan & Wang, Linbo & Zhou, Jichun. (2021). Metformin induces Ferroptosis by inhibiting UFMylation of SLC7A11 in breast cancer. Journal of Experimental & Clinical Cancer Research. 40. 10.1186/s13046-021-02012-7. 

Harada, Makoto & Adam, Jonathan & Covic, Marcela & Ge, Jianhong & Brandmaier, Stefan & Muschet, Caroline & Huang, Jialing & Han, Siyu & Rommel, Martina & Rotter, Markus & Heier, Margit & Mohney, Robert & Krumsiek, Jan & Kastenmüller, Gabi & Rathmann, Wolfgang & Zou, Zhong-Mei & Zukunft, Sven & Scheerer, Markus & Neschen, Susanne & Wang-Sattler, Rui. (2024). Bidirectional modulation of TCA cycle metabolites and anaplerosis by metformin and its combination with SGLT2i. Cardiovascular Diabetology. 23. 10.1186/s12933-024-02288-x. 

Cai L, Jin X, Zhang J, Li L, Zhao J. Metformin suppresses Nrf2-mediated chemoresistance in hepatocellular carcinoma cells by increasing glycolysis. Aging (Albany NY). 2020 Sep 14;12(17):17582-17600. doi: 10.18632/aging.103777. Epub 2020 Sep 14. PMID: 32927432; PMCID: PMC7521529.

Zamanian, Mohammad & Giménez-Llort, Lydia & Nikbakhtzadeh, Marjan & Kamiab, Zahra & Heidari, Mahsa & Bazmandegan, Gholamreza. (2022). The Therapeutic Activities of Metformin: Focus on the Nrf2 Signaling Pathway and Oxidative Stress Amelioration. Current molecular pharmacology. 15. 10.2174/1874467215666220620143655. 

Kocemba-Pilarczyk, Kinga & Trojan, Sonia & Ostrowska, Barbara & Lasota, Małgorzata & Dudzik, Paulina & Kusior, Dorota & Kot, Marta. (2020). Influence of metformin on HIF-1 pathway in multiple myeloma. Pharmacological Reports. 72. 10.1007/s43440-020-00142-x. 

Guimarães, Talita & Farias, Lucyana & Santos, Eliane & Fraga, Carlos Alberto & Orsini, Lissur & Teles, Leandro & Feltenberger, John & Jesus, Sabrina & Souza, Marcela & Santos, Sérgio & Paula, Alfredo & Gomez, Ricardo & Guimaraes, Andre & Guimarã}es, Talita & Farias, Lucyana & Jesus, Sabrina & de Paula, Alfredo & Guimarã}es, André & Alberto, Carlos & Paula, Batista. (2016). Metformin increases PDH and suppresses HIF-1$alpha$ under hypoxic conditions and induces cell death in oral squamous cell carcinoma. Oncotarget. 5. 10.18632/oncotarget.10842. 

Cufí, Silvia & Vazquez-Martin, Alejandro & Oliveras-Ferraros, Cristina & Martín, Begoña & Joven, Jorge & Menendez, Javier. (2010). Metformin against TGFβ-induced epithelial-to-mesenchymal transition (EMT): From cancer stem cells to aging-associated fibrosis. Cell cycle (Georgetown, Tex.). 9. 4461-8. 10.4161/cc.9.22.14048. 

Kim, Jae Hyun & Lee, Kyoung & Seo, Yoojeong & Kwon, Ji-Hee & Yoon, Jae & Kang, Jo & Lee, Hyun & Park, Soo & Hong, Sung & Cheon, Jae & Kim, Won & Kim, Tae. (2018). Effects of metformin on colorectal cancer stem cells depend on alterations in glutamine metabolism. Scientific Reports. 8. 409. 10.1038/s41598-018-29895-5. 

Repurposing Phenylbutyrate: versatile anticancer agent

Phenylbutyrate (PB), a short-chain fatty acid derivative, is primarily recognized as an ammonia scavenger and histone deacetylase inhibitor (HDACi). Initially developed for managing urea cycle disorders, PB has recently gained attention for its anticancer properties. Through its HDAC inhibition, PB can influence gene expression, promoting cell cycle arrest, apoptosis, and differentiation in various cancer cell lines. Beyond its epigenetic effects, PB inhibits critical metabolic enzymes, disrupts cancer cell metabolism, and reduces tumor invasiveness by limiting epithelial–mesenchymal transition (EMT). These multifunctional properties make PB a promising candidate for cancer therapy, either as a standalone agent or combined with conventional treatments, offering new avenues for enhancing therapeutic efficacy against various malignancies.

Histone Deacetylase Inhibition (HDACi)

Phenylbutyrate acts as a histone deacetylase inhibitor, altering gene expression by modifying chromatin structure. This mechanism promotes apoptosis and cell cycle arrest, with studies showing enhanced cytotoxicity in combination with conventional chemotherapy agents in colorectal cancer models. In prostate and multiple myeloma cell lines, phenylbutyrate downregulated anti-apoptotic proteins, such as Bcl-XL, and promoted the pro-apoptotic caspase cascade.

Inhibition of Epithelial–Mesenchymal Transition (EMT)

In oral squamous cell carcinoma (OSCC), phenylbutyrate inhibited EMT by downregulating the transforming growth factor-β (TGF-β) pathway and decreasing mesenchymal markers. This mechanism prevented migration and invasion of OSCC cells, demonstrating significant antitumor effects in vitro and in vivo.

PDK Inhibition and Metabolic Regulation

Phenylbutyrate was found to inhibit specific pyruvate dehydrogenase kinase (PDK) isoforms, especially PDK2 and PDK3, enhancing the pyruvate dehydrogenase complex (PDH) activity. This effect promotes cellular energy production via the tricarboxylic acid (TCA) cycle, potentially disrupting the Warburg effect.

Cell Cycle Arrest and Induction of Differentiation

In glioblastoma and other cancer cell lines, phenylbutyrate induced cell cycle arrest, specifically by upregulating the cell cycle inhibitor p21. Additionally, its differentiation-inducing properties helped reduce the aggressiveness of glioma cells and limit their proliferative potential​.

Apoptosis and Anti-Angiogenesis

Phenylbutyrate enhances apoptosis by downregulating angiogenesis-related factors like vascular endothelial growth factor (VEGF) and caveolin-1. In prostate cancer, it was shown to sensitize cells to radiation therapy, indicating potential synergy with other treatments to inhibit tumor progression.​

Table: Key Mechanisms of Activity 

Phenylbutyrate’s Dual Impact on ROS in Normal vs Malignant Cells

In cancer cells, phenylbutyrate (as a histone deacetylase inhibitor, HDACi) can transiently increase ROS, especially during apoptosis:

• HDAC inhibitors (like phenylbutyrate) selectively increase ROS in tumor cells but not normal ones [ref]
• Sodium butyrate (a close analog) raised ROS and induced apoptosis in breast cancer cells while sparing normal ones [ref]
• Combination treatments with HDAC inhibitors amplify ROS selectively in resistant cancer cells

Phenylbutyrate reduces ROS in normal cells by lowering ER stress and boosting antioxidants, while it transiently increases ROS in malignant cells as part of its pro-apoptotic, anticancer mechanism.

References

Qian, Kun & Sun, Laiyu & Zhou, Guoqing & Ge, Haixia & Meng, Yue & Li, Jingfen & Li, Xiao & Xinqiang, Fang. (2018). Sodium Phenylbutyrate Inhibits Tumor Growth and the Epithelial–Mesenchymal Transition of Oral Squamous Cell Carcinoma In Vitro and In Vivo. Cancer Biotherapy and Radiopharmaceuticals. 33. 10.1089/cbr.2017.2418. 

Homer, Ronald & Rozental, Jack & Duncan, Holly & Engelhard, Herbert. (1998). Inhibitory effects of phenylbutyrate on the proliferation, morphology, migration and invasiveness of malignant glioma cells. Journal of Neuro-Oncology. 37. 97-108. 10.1023/A:1005865125588. 

Ferriero, Rosa & Iannuzzi, Clara & Manco, Giuseppe & Brunetti-Pierri, Nicola. (2015). Differential inhibition of PDKs by phenylbutyrate and enhancement of pyruvate dehydrogenase complex activity by combination with dichloroacetate. Journal of inherited metabolic disease. 38. 10.1007/s10545-014-9808-2. 

Bai, Li-Yuan & Omar, Hany & Chiu, Chang-Fang & Chi, Zeng-Pang & Hu, Jing-Lan & Weng, Jing-Ru. (2010). Antitumor effects of (S)-HDAC42, a phenylbutyrate-derived histone deacetylase inhibitor, in multiple myeloma cells. Cancer chemotherapy and pharmacology. 68. 489-96. 10.1007/s00280-010-1501-z. 

Alkeilani, Maha & Alsmadi, Dua. (2018). THE HDAC INHIBITOR SODIUM PHENYLBUTYRATE ENHANCES THE CYTOTOXICITY INDUCED BY 5-FLUOROURACIL, OXALIPLATIN, AND IRINOTECAN IN COLORECTAL CANCER CELL LINES. International Journal of Pharmacy and Pharmaceutical Sciences. 10. 155. 10.22159/ijpps.2018v10i1.22947. 

Goh, Meidee & Chen, Feng & Paulsen, Michelle & Yeager, Ann & Dyer, Erica & Ljungman, Mats. (2001). Phenylbutyrate attenuates the expression of Bcl-X(L), DNA-PK, caveolin-1, and VEGF in prostate cancer cells. Neoplasia (New York, N.Y.). 3. 331-8. 10.1038/sj/neo/7900165. 

Burzynski, Stanislaw & Patil, Sonali. (2014). The Effect of Antineoplastons A10 and AS2-1 and Metabolites of Sodium Phenylbutyrate on Gene Expression in Glioblastoma Multiforme. Journal of Cancer Therapy. 5. 929-945. 10.4236/jct.2014.510099. 

Repurposing Niclosamide: A Multi-Targeted Approach to Cancer Therapy Through Metabolic Disruption, Immune Modulation, and Oncogenic Pathway Inhibition

Niclosamide, approved initially as an anthelmintic, has garnered significant attention in oncology for its anticancer potential. The repositioning of niclosamide is particularly appealing due to its established safety profile and diverse mechanisms targeting oncogenic pathways, metabolic processes, and immune checkpoints. Key research across a spectrum of cancer types, including lung, colorectal, breast, and hematological cancers, emphasizes its role in disrupting multiple pathways critical for cancer cell survival and proliferation.

Targeting the Wnt/β-Catenin Pathway in Colorectal Cancer (CRC)

One of niclosamide's primary mechanisms in cancer involves inhibiting the Wnt/β-catenin pathway, an oncogenic driver in various cancers, particularly colorectal cancer. The study in CRC showed that niclosamide reduces the expression of S100A4, a metastasis-promoting protein driven by Wnt/β-catenin. This reduction in S100A4 levels suppresses metastatic potential, indicating that niclosamide could improve outcomes for metastatic CRC patients with high Wnt/β-catenin activity.

STAT3 Inhibition and Radiosensitization in Lung Cancer

Another study highlighted niclosamide’s ability to inhibit STAT3, a transcription factor linked to tumor growth and immune evasion, especially in non-small cell lung cancer (NSCLC). The drug blocks STAT3 activation, reversing radioresistance and improving radiotherapy outcomes. Additionally, niclosamide downregulates PD-L1 expression, enhancing the efficacy of immune checkpoint inhibitors like PD-1/PD-L1 antibodies. This dual inhibition of STAT3 and PD-L1 can improve T-cell infiltration and tumor cell lysis, thus presenting a promising combination strategy with immunotherapy for NSCLC.

CREB-Dependent Pathway Suppression in Acute Myeloid Leukemia (AML)

In AML, niclosamide disrupts CREB, a critical cancer cell survival and proliferation regulator. By inhibiting CREB-dependent signaling, niclosamide induces apoptosis and cell cycle arrest in AML cells while sparing normal hematopoietic cells. Preclinical studies further demonstrate niclosamide's potential to synergize with traditional chemotherapeutics, enhancing cytotoxicity and improving treatment response. The effectiveness of niclosamide in targeting CREB suggests its applicability in other CREB-overexpressing cancers as well.

Inducing Oxidative Stress and Ferroptosis in Triple-Negative Breast Cancer (TNBC)

Niclosamide’s ability to induce ferroptosis—a form of programmed cell death reliant on iron and lipid peroxidation—was observed in triple-negative breast cancer cells. By inhibiting the transporters SLC38A5 and SLC7A11, which modulate glutathione levels and antioxidant defenses, niclosamide depletes cellular glutathione, disrupts redox balance, and enhances lipid peroxidation. This oxidative stress-mediated cell death pathway offers a novel therapeutic angle for targeting resistant breast cancer subtypes.

Mitochondrial Uncoupling and Metabolic Reprogramming in Solid Tumors

Niclosamide also acts as a mitochondrial uncoupler, disrupting cancer cell metabolism. This uncoupling effect reverses the Warburg effect—a phenomenon in which cancer cells preferentially utilize glycolysis over oxidative phosphorylation—by enhancing mitochondrial respiration and decreasing glycolysis dependence. This metabolic reprogramming impairs tumor growth and inhibits pathways like NF-κB, mTORC1, and Notch, often upregulated in aggressive cancers. The metabolic disruption induced by niclosamide contributes to its broad-spectrum anticancer effects.

Enhancing PD-1/PD-L1 Immunotherapy in NSCLC

Research in NSCLC suggests that combining niclosamide with PD-1/PD-L1 inhibitors augments immune response by increasing T-cell infiltration and activity. Niclosamide decreases PD-L1 expression in tumor cells via STAT3 inhibition, thus sensitizing tumors to immunotherapy. This combination therapy prolongs survival in preclinical models, indicating the potential for broader clinical application in immune checkpoint inhibitor-resistant cancers.

Table: Primary Mechanisms of Niclosamide in Cancer Therapy

Niclosamide’s capacity to interfere with multiple oncogenic pathways and cellular processes renders it a versatile anticancer agent. Its repurposing in oncology illustrates a promising approach for often treatment-resistant cancers. By targeting pathways such as Wnt/β-catenin, STAT3, and mTORC1 and through mechanisms like mitochondrial uncoupling and ferroptosis induction, niclosamide holds potential as both a monotherapy and an adjuvant to standard therapies. Its synergistic effects with immune checkpoint inhibitors reinforce its adaptability in cancer therapeutics.

References

Burock, Susen & Daum, Severin & Keilholz, Ulrich & Neumann, Konrad & Walther, Wolfgang & Stein, Ulrike. (2018). Phase II trial to investigate the safety and efficacy of orally applied niclosamide in patients with metachronous or sychronous metastases of a colorectal cancer progressing after therapy: The NIKOLO trial. BMC Cancer. 18. 10.1186/s12885-018-4197-9. 

Jiang, Haowen & Li, Albert & Ye, Jiangbin. (2022). The magic bullet: Niclosamide. Frontiers in Oncology. 12. 1004978. 10.3389/fonc.2022.1004978. 

Chae, Hee-Don & Cox, Nick & Dahl, Gary & Lacayo, Norman & Davis, Kara & Cappolicchio, Samanta & Smith, Mark & Sakamoto, Kathleen. (2017). Niclosamide suppresses acute myeloid leukemia cell proliferation through inhibition of CREB-dependent signaling pathways. Oncotarget. 9. 10.18632/oncotarget.23794. 

Mathew, Marilyn & Sivaprakasam, Sathish & Dharmalingam-Nandagopal, Gunadharini & Sennoune, Souad & Nguyen, Nhi & Jaramillo-Martinez, Valeria & Bhutia, Yangzom & Ganapathy, Vadivel. (2024). Induction of Oxidative Stress and Ferroptosis in Triple-Negative Breast Cancer Cells by Niclosamide via Blockade of the Function and Expression of SLC38A5 and SLC7A11. Antioxidants. 13. 291. 10.3390/antiox13030291. 

You, Shuo & Li, Rui & Park, Dongkyoo & Xie, Maohua & Sica, Gabriel & Cao, Ya & Xiao, Zhi-Qiang & Deng, Xingming. (2013). Disruption of STAT3 by Niclosamide Reverses Radioresistance of Human Lung Cancer. Molecular cancer therapeutics. 13. 10.1158/1535-7163.MCT-13-0608. 

Shrivastava, Shweta & Kumar, P. & Jeengar, Manish Kumar & Naidu, Vgm. (2014). T3038 - Inhibition of Wnt/β-catenin Pathway by Niclosamide: A Therapeutic Target for Gastric Cancer. 

Jug, Mario & Laffleur, Flavia & Millotti, Gioconda. (2024). Revisiting Niclosamide Formulation Approaches – a Pathway Toward Drug Repositioning. Drug Design, Development and Therapy. 18. 4153-4182. 10.2147/DDDT.S473178. 

Luo, Fan & Luo, Min & Rong, Qi-Xiang & Zhang, Hong & Chen, Zhen & Wang, Fang & Zhao, Hong-Yun & Fu, Li-Wu. (2019). Niclosamide, an antihelmintic drug, enhances efficacy of PD-1/PD-L1 immune checkpoint blockade in non-small cell lung cancer. Journal for ImmunoTherapy of Cancer. 7. 10.1186/s40425-019-0733-7. 

High Dietary Inorganic Phosphate and Lung Tumorigenesis

Two studies, Jin et al. and Lee et al., provide a detailed look into the role of high dietary inorganic phosphate (Pi) in promoting lung tumorigenesis, revealing both early and long-term effects on cancer progression.

Both studies observed significant lung tumor number and size increases in high-Pi diets. Jin et al. reported that a high-Pi diet (1.0%) markedly increased the occurrence of lung tumors compared to a regular diet (0.5%), with notably larger tumor lesions. Lee et al. observed that tumor progression accelerated within the first two months on a high-Pi diet but slowed after prolonged exposure (four months) due to metabolic adaptations. This suggests Pi initially promotes tumor growth but may lead to a state of tumor quiescence over time.

Both studies demonstrated that high Pi intake amplifies the Akt/mTOR signaling pathway. This pathway is essential for cell growth and survival, and its activation was found to support tumor progression. Enhanced phosphorylation of Akt and subsequent increases in protein translation machinery, such as mTOR and 4E-BP1, were reported as essential for synthesizing proteins necessary for cancer cell growth. Lee et al. added that protein translation was notably stimulated during the early stages of tumor development but was downregulated after prolonged Pi exposure, potentially reflecting an adaptive metabolic response in tumor cells.

High Pi intake spurred cellular proliferation and angiogenesis, which are essential for tumor growth and nutrient supply. Increased expression of cell cycle regulators (e.g., cyclin D3, CDK2) and the angiogenic factor FGF-2 was reported in early-stage tumors, correlating with enhanced tumor growth. However, Lee et al. noted a decrease in these factors at later stages, suggesting a switch to a more stable, less proliferative state.

A significant finding in Lee et al.'s study is the role of autophagy in prolonged Pi exposure. Autophagy, the process of cellular recycling under stress, was upregulated in tumor cells subjected to a high-Pi diet over four months. Markers such as LC3 and ATG5 showed increased expression, indicating that the cells might enter a state of quiescence and survival under nutrient-limited conditions, possibly contributing to long-term resistance to tumor growth.

In both studies, High Pi intake was shown to reduce apoptosis, the programmed cell death crucial for eliminating damaged cells. Reduced mitochondrial pro-apoptotic proteins like Bax and caspase-3 activity were observed, suggesting that high Pi enables cancer cells to evade cell death, thereby supporting persistent tumor growth and possibly contributing to more aggressive cancer behavior.

Lee et al. identified significant changes in lung and liver ion levels, with elevated phosphorus, iron, and calcium concentrations reflecting altered metabolic states in these tissues. An upregulation in the tricarboxylic acid (TCA) cycle components was noted, enhancing ATP production. This metabolic shift likely supports the high energy demand of growing tumors, although it later leads to adaptive changes that reduce tumor progression.

The findings from both studies suggest that high dietary Pi can be a risk factor for lung cancer progression. Given its prevalence in processed foods, these results highlight the importance of monitoring dietary Pi intake, especially in individuals at high risk of cancer. Regulating Pi levels in the diet could potentially slow cancer progression, especially in early-stage lung cancer patients, by limiting the nutrient and signaling support tumors require for rapid growth.

The combined research underscores that a high Pi diet initiates rapid early-stage tumor growth through enhanced signaling and cellular proliferation. Over time, metabolic adaptations, including autophagy and quiescence, emerge, possibly leading to more resilient tumors. These insights suggest the need for further exploration of Pi as a modifiable dietary factor in cancer management and prevention.

So be sure to read labels carefully: check the ingredients list to avoid terms like “sodium phosphate,” “potassium phosphate,” “disodium phosphate,” or any other phosphate compounds.

References

Jin H, Xu CX, Lim HT, Park SJ, Shin JY, Chung YS, Park SC, Chang SH, Youn HJ, Lee KH, Lee YS, Ha YC, Chae CH, Beck GR Jr, Cho MH. High dietary inorganic phosphate increases lung tumorigenesis and alters Akt signaling. Am J Respir Crit Care Med. 2009 Jan 1;179(1):59-68. doi: 10.1164/rccm.200802-306OC. Epub 2008 Oct 10. PMID: 18849498; PMCID: PMC2615662.

Lee, Somin & hu, Kim & Hong, Seong-Ho & Lee, Ah Young & Park, Eun-Jung & Seo, Hwi Won & Chae, Chanhee & Doble, Philip & Bishop, David & Cho, Myung-Haing. (2015). High Inorganic Phosphate Intake Promotes Tumorigenesis at Early Stages in a Mouse Model of Lung Cancer. PloS one. 10. e0135582. 10.1371/journal.pone.0135582. 

Combination of RAS(ON) G12C-selective inhibitors with SHP2 inhibitors to sensitize tumours to immune checkpoint blockade

Lung cancer driven by KRAS mutations, particularly the G12C variant, remains a significant challenge in non-small cell lung cancer (NSCLC) treatment. KRAS mutations promote tumor growth and survival, making them ideal therapeutic targets. Although KRASG12C inhibitors such as adagrasib and sotorasib, which target the inactive, GDP-bound form of KRAS, have shown initial success, their efficacy is limited by the rapid development of resistance in many patients.

To address this issue, the authors of this study explored combining RMC-4998, a selective inhibitor targeting the active, GTP-bound form of KRASG12C, with RMC-4550, an SHP2 inhibitor. SHP2 mediates signaling from receptor tyrosine kinases (RTKs), contributing to the reactivation of the RAS pathway even when KRASG12C inhibitors are applied. This dual approach aims to suppress KRAS signaling at multiple levels and remodel the tumor microenvironment (TME) to enhance immune responses.

In cell studies, RMC-4998 significantly outperformed traditional KRAS inhibitors by rapidly reducing KRASG12C-mutant NSCLC cell viability and inhibiting MAPK signaling. However, ERK phosphorylation, a marker of pathway reactivation, rebounded after 24-48 hours, indicating adaptive resistance. When RMC-4550 was added, the rebound effect was suppressed, leading to sustained MAPK inhibition, more significant reductions in cell viability, and increased apoptosis. These effects were observed over long-term treatment, demonstrating that this combination prevents the development of adaptive resistance.

In mouse models of KRASG12C-mutant lung cancer, the combination therapy showed remarkable efficacy. In immunocompetent mice with immunogenic tumors, the dual treatment led to complete tumor regressions in 75% of cases treated with RMC-4998 and in 14.3% with RMC-4550 alone. Significantly, the combination therapy prevented tumor relapse in 100% of treated mice, resulting in durable tumor eradication. Moreover, the combination induced immune memory, as mice remained tumor-free after being rechallenged with cancer cells, showing that the treatment had stimulated lasting anti-tumor immunity.

In more aggressive, immune-excluded tumor models, which typically resist immune checkpoint blockade (ICB) therapies, the combination therapy sensitized tumors to anti-PD-1 treatment. When combined with anti-PD-1 therapy, the treatment resulted in 37.5% complete tumor regressions in these resistant tumors, compared to marginal responses with ICB alone or either RMC-4998 or RMC-4550 monotherapy. The combination of RMC-4998 and RMC-4550 not only slowed tumor growth but also reprogrammed the TME, increasing CD8+ T cell infiltration and suppressing immunosuppressive myeloid cells.

In an orthotopic model of lung cancer, where tumors are located in their natural tissue environment, RMC-4998 treatment resulted in significant tumor shrinkage, with over half of the tumors regressing completely. However, relapse occurred in about 50% of these cases after treatment cessation. When RMC-4550 was added, relapse rates dropped to just 10%, highlighting the benefit of combining both inhibitors in preventing early tumor recurrence.

These preclinical results suggest that combining KRAS(ON) G12C-selective inhibitors with SHP2 inhibition has the potential to dramatically enhance treatment outcomes in KRAS-mutant lung cancers, especially those resistant to standard therapies. The combination not only targets cancer cell survival but also promotes immune responses, offering a promising strategy for achieving durable tumor regressions and improved patient survival. 

References

Anastasiou, P., Moore, C., Rana, S. et al. Combining RAS(ON) G12C-selective inhibitor with SHP2 inhibition sensitises lung tumours to immune checkpoint blockade. Nat Commun 15, 8146 (2024). https://doi.org/10.1038/s41467-024-52324-3

Itraconazole as a Promising Anti-Cancer Agent: Insights from Recent Studies

Itraconazole, a widely used antifungal medication, has emerged as a potential repurposed drug for cancer treatment. Beyond its antifungal properties, itraconazole has effectively inhibited cancer cell proliferation, migration, and survival through various molecular mechanisms. A growing body of research highlights its utility against several cancer types, including ovarian, colon, pancreatic, melanoma, and triple-negative breast cancer (TNBC). Below is a summary of findings from recent studies investigating the anti-cancer effects of itraconazole.

1. Itraconazole in Refractory Ovarian Cancer

A study explored the use of itraconazole in combination with chemotherapy in patients with refractory ovarian cancer. The study involved 55 patients divided into two groups: 19 received itraconazole with chemotherapy, while 36 received chemotherapy alone. Results showed that patients treated with itraconazole had significantly improved progression-free survival (PFS) of 103 days compared to 53 days in the control group, and overall survival (OS) was 642 days versus 139 days, respectively. Itraconazole likely exerts these effects by inhibiting P-glycoprotein, a protein associated with drug resistance, and reducing angiogenesis. These findings suggest that itraconazole could be a valuable adjunct therapy in the management of platinum-resistant ovarian cancer.

2. Itraconazole in Colon Cancer

A study involving 5,221 patients with colon cancer, based on the Taiwanese National Health Insurance Research Database, demonstrated that itraconazole improved the 5-year survival rate in patients with advanced-stage colon cancer (stages III and IV). In vitro experiments showed that itraconazole inhibited colon cancer cell proliferation, induced apoptosis, and caused cell cycle arrest at the G1 phase. Additionally, itraconazole triggered autophagic cell death and inhibited the transketolase (TKT) enzyme, a key player in cancer metabolism. These results underscore the potential of itraconazole as a repurposed therapy for colon cancer, particularly in combination with chemotherapy.

3. Itraconazole in Pancreatic Cancer

In a study focusing on pancreatic cancer, itraconazole was found to inhibit the invasion and migration of pancreatic cancer cells by suppressing the TGF-β/SMAD2/3 signaling pathway, a critical pathway involved in epithelial-to-mesenchymal transition (EMT) and metastasis. The drug also reversed EMT by increasing E-cadherin expression and decreasing N-cadherin and vimentin levels, markers associated with cancer cell migration. In vivo studies in a transgenic mouse model showed that itraconazole significantly reduced tumor growth and inhibited key markers of cancer progression. These findings suggest that itraconazole could be a valuable agent in limiting pancreatic cancer metastasis.

4. Itraconazole in Triple-Negative Breast Cancer (TNBC)

The combination of itraconazole and rapamycin was investigated as a treatment for triple-negative breast cancer (TNBC). The study found that the combination exerted synergistic effects, significantly inhibiting TNBC cell proliferation and migration. This combination led to cell cycle arrest at the G0/G1 phase and suppressed the AKT/mTOR signaling pathway, which is crucial for cancer cell survival. Although the combination did not induce significant apoptosis, its ability to halt cell cycle progression highlights the potential of itraconazole in treating TNBC when combined with other therapies.

5. Itraconazole in Melanoma

In a study targeting melanoma, itraconazole inhibited tumor growth by suppressing the Hedgehog (Hh), Wnt, and PI3K/mTOR signaling pathways. In vitro experiments revealed that itraconazole significantly reduced the proliferation of melanoma cells and colony formation, and it induced downregulation of key markers such as Gli-1, Gli-2, Wnt3A, and β-catenin. The drug also increased Gli-3 and Axin-1, which act as repressors of the Hh and Wnt pathways. In a mouse model, itraconazole reduced tumor volume, extended survival, and inhibited cell proliferation markers like Ki-67. These findings suggest that itraconazole could effectively treat melanoma by disrupting multiple oncogenic pathways.

The accumulating evidence on itraconazole's anti-cancer properties across various cancer types (including ovarian, colon, pancreatic, TNBC, and melanoma) demonstrates its potential as an effective repurposed drug. Its ability to inhibit essential cancer signaling pathways such as PI3K/mTOR, Hedgehog, and Wnt, along with its impact on cell cycle arrest, motility, apoptosis, and autophagy, makes itraconazole a promising candidate for combination therapies aimed at improving cancer treatment outcomes.

Combining azoles with other substances can enhance activity, reduce resistance, and lower toxicity. Various existing drugs, such as statins, bisphosphonates, and immunomodulators, show potential for enhancing azole treatments. Statins, for instance, can synergize with azoles. Many plant-based compounds, such as thymol, carvacrol, and berberine, demonstrate strong synergy with azoles, showing promise as adjuvants.

References

Chen, Ke & Cheng, Liang & Qian, Weikun & Jiang, Zhengdong & Sun, Liankang & Zhao, Yanfei & Zhou, Yongsheng & Zhao, Lizhi & Wang, Pengli & Duan, Wanxing & Ma, Qingyong & Yang, Wei. (2018). Itraconazole inhibits invasion and migration of pancreatic cancer cells by suppressing TGF-β/SMAD2/3 signaling. Oncology Reports. 39. 10.3892/or.2018.6281. 

Wu, Hua-Tao & Li, Chun-Lan & Fang, Ze-Xuan & Chen, Wen-Jia & Lin, Wen-Ting & Liu, Jing. (2022). Induced Cell Cycle Arrest in Triple-Negative Breast Cancer by Combined Treatment of Itraconazole and Rapamycin. Frontiers in Pharmacology. 13. 873131. 10.3389/fphar.2022.873131. 

Xu, Congcong & Zhuo, Yating & Liu, Yunyao & Chen, Hao. (2022). Itraconazole Inhibits the Growth of Cutaneous Squamous Cell Carcinoma by Targeting HMGCS1/ACSL4 Axis. Frontiers in Pharmacology. 13. 10.3389/fphar.2022.828983. 

Shen, Pei-Wen & Chou, Yu-Mei & Li, Chia-Ling & Liao, En-Chih & Huang, Hung-Sen & Yin, Chun-Hao & Chen, Chien-liang & Yu, Sheng-Jie. (2021). Itraconazole improves survival outcomes in patients with colon cancer by inducing autophagic cell death and inhibiting transketolase expression. Oncology Letters. 22. 10.3892/ol.2021.13029. 

Tsubamoto, Hiroshi & Sonoda, Takashi & Yamasaki, Masaaki & Inoue, Kayo. (2014). Impact of Combination Chemotherapy with Itraconazole on Survival of Patients with Refractory Ovarian Cancer. Anticancer research. 34. 2481-7. 

Kane A, Carter DA. Augmenting Azoles with Drug Synergy to Expand the Antifungal Toolbox. Pharmaceuticals (Basel). 2022 Apr 14;15(4):482. doi: 10.3390/ph15040482. PMID: 35455479; PMCID: PMC9027798.Kane A, Carter DA. Augmenting Azoles with Drug Synergy to Expand the Antifungal Toolbox. Pharmaceuticals (Basel). 2022 Apr 14;15(4):482. doi: 10.3390/ph15040482. PMID: 35455479; PMCID: PMC9027798.

Lin MC, Chuang YT, Wu HY, Hsu CL, Lin NY, Huang MC, Lou PJ. Targeting tumor O-glycosylation modulates cancer-immune-cell crosstalk and enhances anti-PD-1 immunotherapy in head and neck cancer. Mol Oncol. 2024 Feb;18(2):350-368. doi: 10.1002/1878-0261.13489. Epub 2023 Jul 24. PMID: 37452653; PMCID: PMC10850803.

Zhang, Yong & Li, Lu & Chu, Feifei & Wu, Huili & Xiao, Xingguo & Ye, Jianping & Li, Kunkun. (2024). Itraconazole inhibits tumor growth via CEBPB ‐mediated glycolysis in colorectal cancer. Cancer Science. 115. 10.1111/cas.16082. 

Fasting and Silibinin: A Synergistic Approach to Tumor Suppression

Fasting has long been recognized as a natural strategy for enhancing health, with numerous studies demonstrating its benefits for inhibiting cancer progression. However, prolonged fasting can pose challenges, particularly for cancer patients who may already be in a weakened state. Enter silibinin—a natural polyphenolic compound extracted from Silybum marianum (milk thistle), which has gained attention for its ability to mimic the effects of fasting. Silibinin exhibits potent anti-tumor properties, and when combined with fasting, the effects on cancer suppression can be magnified. This article explores how fasting and silibinin work together to inhibit hepatocellular carcinoma (HCC) and other cancers while estimating the potential tumor reduction when combining the two approaches.

Fasting has been shown to suppress cancer progression through several mechanisms. One of its primary effects is the inhibition of glycolysis, the process cancer cells rely on for energy. By depleting glucose availability and triggering metabolic stress, fasting selectively affects cancer cells more than normal cells. Cancer cells, highly dependent on glucose, struggle to adapt to these energy deficits. Additionally, fasting enhances anti-cancer immune responses and induces autophagy, a cellular recycling process that can promote the death of damaged or cancerous cells.

Studies have demonstrated that fasting alone can significantly reduce tumor size. For instance, a study involving calorie restriction in a rat model of liver cancer showed an 85% reduction in preneoplastic liver lesions after a prolonged fasting regimen.

Silibinin has garnered attention for its hepatoprotective properties, particularly its ability to act as a fasting mimetic. It exerts anti-tumor effects through multiple mechanisms, including activating AMP-activated protein kinase (AMPK), a key energy sensor that mimics the metabolic stress induced by fasting. This compound inhibits the glycolytic process, reduces glucose uptake, and decreases intracellular ATP levels, further enhancing cancer cell vulnerability.

In hepatocellular carcinoma (HCC) models, silibinin has been shown to upregulate death receptor 5 (DR5) through AMPK activation, leading to extrinsic apoptosis—programmed cell death driven by external signals. Additionally, silibinin weakens cancer cells' metabolic foundation by inhibiting glycolysis and reducing glucose availability.

While fasting and silibinin independently exhibit strong anti-cancer effects, their combination produces a synergistic effect that significantly amplifies tumor suppression. Fasting and silibinin activate the AMPK pathway, leading to a coordinated depletion of energy resources in cancer cells and triggering their death. Moreover, fasting-induced stress sensitizes cancer cells to silibinin, allowing for more efficient elimination of malignant cells.

In vivo studies using xenograft mouse models of HCC, fasting, and silibinin led to remarkable tumor size reductions. While silibinin alone (at 400 mg/kg/day) significantly suppressed tumor growth, fasting further decreased tumor size and weight by approximately 25% more than silibinin alone. The dual approach proved particularly effective at reducing tumor weight while causing minimal side effects, as fasting-induced weight loss in the animal models returned to baseline levels after refeeding periods.

A key mechanism underlying the synergistic effect of fasting and silibinin is the activation of AMPK, which plays a critical role in cellular energy homeostasis. AMPK activation, triggered by fasting and silibinin, leads to the phosphorylation of downstream targets like ULK1, which promotes autophagy and inhibits tumor cell survival. Moreover, silibinin-induced AMPK activation upregulates DR5, leading to extrinsic apoptosis in cancer cells.

This mechanism highlights the ability of silibinin to mimic fasting by depleting cancer cells of energy and activating apoptotic pathways, resulting in significant tumor suppression without harming normal cells.

Based on the current research in mouse models, it is estimated that silibinin alone can reduce tumor size in HCC models by approximately 40%. However, when combined with fasting, this reduction is enhanced, resulting in a total tumor size decrease of 60-70%. This synergy stems from the overlapping yet distinct pathways through which fasting and silibinin exert their effects—fasting primarily through metabolic stress and silibinin through AMPK activation and apoptosis induction.

The combination of fasting and silibinin offers a promising strategy for cancer treatment, particularly for patients with HCC. By mimicking the metabolic stress of fasting and triggering extrinsic apoptosis, silibinin enhances the anti-tumor effects of fasting, leading to substantial tumor suppression. While more clinical trials are needed to confirm these results in humans, the preclinical data strongly support silibinin as a fasting mimetic and an adjunct therapy in cancer treatment.

References

Xiao, Biying & Jiang, Yanyu & Yuan, Shuying & Cai, Lili & Xu, Tong & Jia, Lijun. (2024). Silibinin, a potential fasting mimetic, inhibits hepatocellular carcinoma by triggering extrinsic apoptosis. MedComm. 5. e457. 10.1002/mco2.457. 

AHCC® Promotes the Anti-Tumor Effect of Dual Immune Checkpoint Blockade Effect in Murine Colon Cancer

The study titled "AHCC®, a Standardized Extract of Cultured Lentinula Edodes Mycelia, Promotes the Anti-Tumor Effect of Dual Immune Checkpoint Blockade Effect in Murine Colon Cancer" by Hong-Jai Park et al. was published in Frontiers in Immunology in April 2022. It explores the potential of Active Hexose Correlated Compound (AHCC®), a mushroom-derived supplement, to enhance the efficacy of dual immune checkpoint blockade (DICB) in a murine model of colon cancer.

Checkpoint inhibitors targeting proteins such as PD-1 and CTLA-4 have revolutionized cancer treatment by restoring T-cell function and allowing the immune system to attack tumors more effectively. However, many cancers either do not respond to these treatments or develop resistance, while patients may experience immune-related severe side effects. As a result, researchers have been exploring combination therapies, including natural compounds like AHCC®, to improve treatment outcomes.

AHCC® is a standardized extract from the mycelia of Lentinula edodes (shiitake mushrooms). It has been recognized for its immune-enhancing properties, particularly in supporting T-cell and natural killer (NK) cell function. AHCC® is composed mainly of oligosaccharides, amino acids, and minerals and has been studied for its anti-cancer potential in animal models. In this study, the authors sought to investigate whether AHCC® could augment the anti-tumor effect of DICB therapy by examining tumor growth inhibition, immune cell activity, and gut microbiota changes.

In the study, C57BL/6 mice were subcutaneously inoculated with MC38 colon carcinoma cells. Three days after tumor inoculation, the mice were treated with either water or AHCC® (18 mg/mouse/day). Additionally, the treatment groups received dual immune checkpoint blockade, with anti-PD-1 (50 µg/mouse) and anti-CTLA-4 (50 µg/mouse) antibodies administered intraperitoneally twice at 3-day intervals once the tumors reached 50–100 mm³.

The combination of AHCC® and DICB significantly reduced tumor volumes compared to water and DICB. The mice treated with AHCC® and DICB exhibited smaller tumor sizes throughout the experiment, indicating an enhanced tumor-suppressive effect. Although the study does not provide specific percentages of tumor inhibition, the data suggest that AHCC® contributed to a marked tumor growth inhibition when combined with immune checkpoint therapy. This finding demonstrates the additive potential of AHCC® in improving the efficacy of immunotherapy.

The researchers further explored the impact of AHCC® on the immune response by examining the activity of tumor-infiltrating lymphocytes. Flow cytometric analysis revealed that mice treated with AHCC® and DICB exhibited increased granzyme B and Ki-67 expression in CD8+ T cells compared to those treated with water and DICB. Granzyme B is a critical cytotoxic molecule involved in T-cell-mediated tumor killing, while Ki-67 is a cell proliferation marker. These results suggest that the enhanced tumor suppression observed in the AHCC®-treated group was partly due to improved T-cell activation, proliferation, and cytotoxicity.

Interestingly, the combination treatment also affected CD4+ T cells, with increased Ki-67 expression and decreased PD-1 expression in tumor-infiltrating CD4+ T cells. The changes in CD8+ and CD4+ T cells in the AHCC®-treated mice indicate that the compound may enhance the cytotoxic and helper T-cell responses in the tumor microenvironment.

In addition to modulating immune cell activity, AHCC® appeared to influence the gut microbiome composition in treated mice. Fecal samples from the mice were analyzed for bacterial diversity, revealing an increased abundance of bacterial species from the Ruminococcaceae family in the AHCC® plus DICB group. This finding is significant because certain members of the Ruminococcaceae family have been linked to better responses to cancer immunotherapy. To further support this connection, the researchers treated some mice with antibiotics to deplete their gut microbiota. In these mice, the addition of AHCC® no longer conferred any therapeutic benefit, suggesting that the gut microbiome plays a crucial role in mediating the enhanced anti-tumor effects of AHCC®.

This study shows the potential of AHCC® as an effective adjuvant to immune checkpoint blockade therapy. The combination of AHCC® and DICB resulted in significant tumor growth inhibition, likely due to enhanced T-cell activation and changes in the gut microbiota. The study underscores the role of the gut microbiome in cancer treatment efficacy, suggesting that future studies should focus on identifying specific bacterial species involved in this response.

References

Park HJ, Boo S, Park I, Shin MS, Takahashi T, Takanari J, Homma K, Kang I. AHCC®, a Standardized Extract of Cultured Lentinula Edodes Mycelia, Promotes the Anti-Tumor Effect of Dual Immune Checkpoint Blockade Effect in Murine Colon Cancer. Front Immunol. 2022 Apr 20;13:875872. doi: 10.3389/fimmu.2022.875872. PMID: 35514996; PMCID: PMC9066372.

Ammonia use in household products: the smoking gun?

Ammonia use in household products like cleaning agents has declined over the years due to safety concerns and the rise of eco-friendly alternatives. While there isn't specific historical data available on the exact usage of ammonia in household products over the last 50 years, this line graph illustrates the trend based on known factors such as increased awareness of chemical safety, the rise of green alternatives, and shifts in cleaning product formulations. This can represent a general idea of how ammonia use may have fluctuated over time.

The following graph shows the trend of ammonia use in household products and lung cancer incidence over the last 50 years. The ammonia use trend is represented by a solid blue line, while lung cancer incidence is shown by a dashed red line:

The third graph includes another line (green) representing the evolution of smokers over the same period.

Ammonia is often added to cigarettes. It is used as part of the manufacturing process to enhance nicotine absorption. Ammonia works by altering the pH level of the tobacco, which allows nicotine to be absorbed more quickly into the bloodstream, making cigarettes more addictive. This technique is sometimes referred to as "freebasing" nicotine, similar to how certain drugs are processed to increase their potency.

Correlation doesn't imply causation, but it is striking nonetheless.

Why Are Women Who Never Smoked Getting Lung Cancer? Women make up about two-thirds of lung cancer cases in never-smokers. {article}

A wide range of commercial cleaning products contains ammonia, such as window and glass cleaners, furniture polish, all-purpose cleaners, stainless-less steel cleaners, floor polishing waxes, toilet cleaners, and oven cleaners. {ref}