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 (low dose) 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.

Caution: High Dose Metformin and AMPK Activation: Transient vs. Chronic Effects on Metastasis

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. 

Wang S, Lin Y, Zhao Q, Chen H, Du S, Zeng Z. Metformin reverses 5-FU resistance induced by radiotherapy through mediating folate metabolism in colorectal cancer. Mol Med. 2025 May 21;31(1):199. doi: 10.1186/s10020-025-01206-5. PMID: 40399808; PMCID: PMC12093704.

Repurposing Phenylbutyrate: versatile anticancer agent

Sodium Phenylbutyrate: Redefining Therapeutic Potential Through Epigenetic Modulation

From Urea Cycle Disorders to Cancer: FDA-Approved Agent Achieves Complete Tumor Regression

Sodium phenylbutyrate emerges as a uniquely positioned anticancer agent, requiring millimolar concentrations 100-1000 times higher than modern HDAC inhibitors yet achieving complete tumor regression in recurrent malignant glioma through monotherapy. This FDA-approved compound exploits cancer cells' epigenetic vulnerabilities through histone deacetylase inhibition, metabolic reprogramming via PDK inhibition, ammonia and glutamine depletion, and selective oxidative stress modulation while maintaining exceptional safety profiles enabling sustained therapeutic exposure impossible with more potent agents.

Sodium Phenylbutyrate

The Moffitt Miracle: Complete Tumor Regression in Recurrent Glioma

The extraordinary case reported by Baker and colleagues at H. Lee Moffitt Cancer Center fundamentally challenged conventional neuro-oncology understanding. A 44-year-old female with recurrent, multicentric malignant glioma having failed radiation, PCV chemotherapy, and four cycles of BCNU/cisplatinum achieved complete tumor regression with phenylbutyrate monotherapy alone.

Historical Significance: Starting at 18g daily in three divided doses, reduced sequentially to 9g then 4.5g due to mild reversible side effects, the patient demonstrated complete radiographic response after nine months. The remission persisted beyond four years with continued low-dose maintenance therapy, achieving a Karnofsky Performance Status score of 100%. This outcome proved historically unprecedented. Within the NABTT CNS Consortium Phase I trial of 23 patients, only this single patient achieved complete response.

This case establishes clinical proof-of-concept that phenylbutyrate can achieve extraordinary outcomes despite modest in vitro potency. The rarity of complete tumor regression using any single non-cytotoxic agent in recurrent high-grade gliomas underscores the profound significance of this clinical observation. Subsequent combination studies amplify this promise: phenylbutyrate with pazopanib, everolimus, and bevacizumab achieved 54.5% objective response rates in recurrent glioblastoma.

Bioavailability-Adjusted Feasibility Comparison

The following comprehensive analysis compares anticancer compounds based on their real-world clinical feasibility, incorporating pharmacokinetic data and achievable plasma concentrations. This table demonstrates why bioavailability often trumps raw potency for practical therapeutic applications.

Compound	Avg IC50 (μM)	Typical Dose	Achievable Cmax (μM)	Ratio	Feasibility Notes
Phenylbutyrate	~4500	18-27g/day (oral, cancer trials)	~1225	~0.27	High; exceeds therapeutic threshold (>500 μM) for ~3h; well-tolerated for sustained use, bioavailability 78%.
Artemisinin and Derivatives	~76 (parent) ~10 (DHA/artesunate)	Oral: 200-400 mg/day IV: 18 mg/kg	Oral: ~3.4 IV: ~83	~0.3 (oral) ~8 (IV)	High; oral limited (bioavailability ~30%), but IV achieves levels for lower IC50s; short half-life (~0.5h) requires frequent dosing.
Shikonin	~5	Limited human data Animal: 10-25 mg/kg	~2-3 (rat extrapolation)	~0.4-0.6	Moderate; low oral bioavailability; nano-formulations needed to improve; promising if levels sustained, but scarce human PK.
Ivermectin	~6	0.2-2 mg/kg	~0.28 (at 2 mg/kg) ~0.1 (at safer 0.4 mg/kg)	~0.05	Low; Cmax far below IC50, though some effects at 0.01-1 μM; food increases by 2.5x; higher doses tolerated but untested for cancer long-term.
Curcumin	~38	4-8 g/day (standard) Formulations: 0.5-3 g	<0 .01="" br="" free="" standard="">~0.35 (formulated)	<0 .001="" br="" standard="">~0.01 (formulated)	Low; mostly conjugated (inactive); micelles/piperine boost to 1-3 μM, but still below IC50; potential via metabolites or tissue accumulation.

This analysis reveals that phenylbutyrate's sustained therapeutic concentrations (1.2 mM) combined with its excellent safety profile create practical advantages over compounds with superior in vitro potency but poor bioavailability. Artemisinin IV shows the highest achievability ratio but requires specialized administration, while curcumin's impressive research profile fails to translate clinically without significant formulation enhancements.

Primary Anticancer Mechanisms

Phenylbutyrate's anticancer activity operates through multiple interconnected mechanisms that distinguish it from conventional chemotherapies and other repurposed agents. The compound exploits cancer cells' epigenetic vulnerabilities while simultaneously targeting metabolic dependencies and inducing selective oxidative stress.

Mechanism	Description
Histone Deacetylase Inhibition (HDACi)	Alters gene expression, inducing apoptosis and cell cycle arrest; enhances the efficacy of chemotherapy in combination settings.
Inhibition of Epithelial-Mesenchymal Transition (EMT)	Downregulates TGF-β signaling, reducing cancer cell invasion and migration, specifically in OSCC.
PDK Inhibition and Metabolic Regulation	Inhibits specific PDK isoforms, promoting PDH activity and altering cancer cell metabolism, which disrupts the Warburg effect.
Cell Cycle Arrest and Induction of Differentiation	Upregulates p21, induces cell differentiation, and limits cancer cell proliferation, particularly effective in glioma and prostate cancer models.
Apoptosis and Anti-Angiogenesis	Promotes caspase activation, decreases anti-apoptotic proteins, and downregulates VEGF, enhancing apoptosis and reducing tumor vascularization.
Selective Oxidative Stress Modulation	Reduces ROS in normal cells while transiently increasing ROS in malignant cells as part of its pro-apoptotic mechanism.
Radiosensitization	Enhances radiation therapy effectiveness with enhancement ratios of 1.3-1.5, particularly in p53-mutant glioblastoma cells.
Combination Synergy	Demonstrates synergistic effects with cisplatin (>1.6x), chemotherapy agents, and targeted therapies while providing cardioprotection against doxorubicin.

Critical Consideration: IL-8 Modulation and Combination Strategies

Recent research reveals that phenylbutyrate's HDAC inhibition can have cancer-type-specific effects on IL-8 (CXCL8) expression, creating both therapeutic opportunities and potential complications. In certain cancers, phenylbutyrate upregulates IL-8, which may promote tumor migration and invasion, potentially counteracting its beneficial effects. Understanding these context-dependent responses is crucial for optimizing combination strategies.

Study/Cancer Type	Phenylbutyrate Effect on IL-8	Main Mechanism	Implications for Combination with IL-8 Inhibitors
Gastric Cancer (MGC-803, BGC-823 cells)	Upregulation (mRNA and secretion increased)	HDAC inhibition → H3 acetylation at IL-8 promoter → Gab2-ERK activation → EMT/migration	Block IL-8 to prevent pro-tumor migration; combine with ERK/Gab2 inhibitors for synergy
Bladder Cancer (UMUC1, 5637, J82 cells)	Downregulation (in monocytes and co-cultures)	HDAC inhibition → reduced M2 polarization and cytokine production	May not require IL-8 inhibition, but enhances TME remodeling; synergize with ICIs via PD-L1 upregulation
Ovarian Cancer (SKOV3, OVCAR3 cells)	Upregulation (via class I HDACi)	IKK-NFκB p65 acetylation → promoter recruitment; CBP-dependent	Inhibit IL-8 or IKK to limit MDSC recruitment and improve HDACi efficacy in solid tumors
Inflammation Models (Ocular/Skin)	Downregulation (with TNF-α, IL-6)	NF-κB antagonism	Supports anti-inflammatory role; combination useful in IL-8-high inflammatory cancers
NSCLC (A549, Calu1 cells)	No direct IL-8 effect reported	Synergy with chemo/TKIs via gene reprogramming	Potential to add IL-8 inhibition if induction occurs, enhancing resistance reversal

Strategic Implication: These findings suggest that phenylbutyrate's therapeutic window could be significantly enhanced through rational combination with IL-8 inhibitors or pathway modulators, in particluar for gastric and ovarian cancers where IL-8 upregulation occurs. Combining phenylbutyrate with IL-8 inhibitors could block potential adverse IL-8-mediated effects, enhancing net anti-tumor activity.

Epigenetic Reprogramming Through HDAC Inhibition

Phenylbutyrate functions as a pan-HDAC inhibitor targeting multiple isoforms across Class I and Class II families. Despite millimolar IC50 requirements, the compound achieves profound epigenetic effects through sustained exposure impossible with nanomolar-potent inhibitors. p21WAF1/CIP1 expression increases 521%, while interleukin-6 rises 603%, demonstrating robust transcriptional modulation at clinically achievable concentrations.

Metabolic Reprogramming Reverses Warburg Phenotype

Unique among HDAC inhibitors, phenylbutyrate inhibits pyruvate dehydrogenase kinases, forcing cancer cells from aerobic glycolysis toward oxidative phosphorylation. This metabolic shift proves devastating for glycolysis-dependent tumors while sparing metabolically flexible normal cells. Lactate production decreases markedly while oxygen consumption increases, confirming successful metabolic reprogramming.

Cancer Type Specificity and Clinical Applications

Phenylbutyrate demonstrates activity across diverse malignancies with cancer-specific response patterns that inform optimal clinical applications. The compound shows particular promise in brain tumors, where its ability to cross the blood-brain barrier provides therapeutic access unavailable to many agents.

Glioblastoma Excellence: Beyond the Moffitt case and this case report, recent combination studies achieved 54.5% objective response rates (18.2% complete response, 36.3% partial response) in recurrent glioblastoma. Enhancement ratios of 1.3-1.5 with radiation therapy demonstrate consistent radiosensitization effects. Pancreatic cancer patients receiving phenylbutyrate combinations achieved 10.5 months median overall survival versus 2.9-6.5 months with other second-line therapies, a two-to-three-fold survival improvement.

Hematologic Malignancies

In acute myeloid leukemia and myelodysplastic syndromes, maximum tolerated doses reach 375 mg/kg/day via continuous infusion, achieving plasma concentrations of 0.29 ± 0.16 mM. Four of 27 patients demonstrated hematological improvement in Phase I trials. Multiple myeloma shows differentiation and growth inhibition, with AR-42, a phenylbutyrate derivative, demonstrating enhanced activity.

Solid Tumor Applications

Non-small cell lung cancer demonstrates synergy values exceeding 1.6 when combined with cisplatin, erlotinib, or gefitinib. Head and neck cancers show particular sensitivity through Fanconi anemia pathway interference. Colorectal cancer responds at 5-10 mM concentrations with enhanced effects when combined with 5-fluorouracil, irinotecan, or oxaliplatin.

Pharmacokinetics and Clinical Dosing

Phenylbutyrate achieves 78% oral bioavailability with rapid absorption reaching peak plasma concentrations within one hour. The compound readily crosses the blood-brain barrier, achieving therapeutic CNS concentrations confirmed in primate studies. Maximum tolerated doses reach 27g daily for solid tumors, maintaining plasma concentrations exceeding 1 mM.

Dosing and Tolerability Profile:

Maximum Tolerated Dose: 27g daily (solid tumors)
Therapeutic Concentrations: 706 μM (9g/day), 1,225 μM (27g/day)
Common Side Effects: GI symptoms, mild neurocognitive effects, body odor
Serious Toxicities: Rare hypocalcemia, hypokalemia with monitoring
Formulations: Buphenyl (sodium salt), Ravicti (glycerol prodrug)

Advanced Delivery Approaches

Novel formulations address current limitations. AN-113, a phenylbutyrate prodrug, demonstrates 20-fold enhanced potency while maintaining safety advantages. Glycerol phenylbutyrate (Ravicti) offers nearly tasteless administration with more sustained drug exposure. These innovations could transform phenylbutyrate from a modestly effective agent into a cornerstone of combination cancer therapy.

Combination Strategies and Synergistic Effects

Phenylbutyrate's true potential emerges through rational combinations exploiting distinct mechanisms. With temozolomide in glioblastoma, the combination induces pronounced autophagic cell death. Radiosensitization proves consistent with enhancement ratios of 1.3-1.5 in p53-mutant cells. Chemotherapy combinations leverage phenylbutyrate's ability to enhance drug uptake and retention.

Remarkable Cardioprotection: Phenylbutyrate provides cardioprotection against doxorubicin, reducing adriamycin-induced cardiac toxicity by over 70% through manganese superoxide dismutase upregulation. This protective effect enables higher chemotherapy doses while safeguarding normal tissues transforming the risk-benefit equation for combination therapy.

Radiation Therapy Enhancement

Mechanistically, phenylbutyrate prevents radiation-induced DNA repair through p21-independent cytostasis and sustained histone hyperacetylation. The compound sensitizes glioblastoma cells lacking wild-type p53 function to ionizing radiation, expanding treatment options for typically radioresistant tumors. Enhancement effects persist across multiple cancer types, suggesting broad applicability.

Phenylbutyrate and Vitamin D3 Synergize Against Cancer

Both agents independently can inhibit pathways like NF-κB and STAT3, which are critical for cancer cell survival, proliferation, and inflammation. Their combination leads to a more potent and sustained suppression of these pathways. Also, the combination can shift the balance from immunosuppressive Regulatory T-cells (Tregs) towards effector T-cells (CD4+ Helper and CD8+ Cytotoxic T-cells).

Clinical Development Status and Future Directions

Despite decades of investigation including multiple Phase I/II trials, phenylbutyrate lacks Phase III validation for cancer indications. However, its established maximum tolerated dose and extensive safety data from urea cycle disorder treatment provide clear development pathways. Off-label use continues expanding as clinicians recognize phenylbutyrate's potential, particularly for brain tumors where treatment options remain limited.

Strategic Development Priorities

• Biomarker-Driven Selection: p53 status affects radiosensitization, MGMT methylation might predict temozolomide synergy
• Combination Protocols: Leveraging demonstrated synergy with radiation, chemotherapy, and targeted agents
• Pediatric Applications: Established pediatric safety data enable investigation in childhood cancers
• Novel Formulations: Prodrugs and nanoformulations addressing current limitations

Key Research Citations

1. Chang, S.M. et al. Oral sodium phenylbutyrate in patients with recurrent malignant gliomas: A dose escalation and pharmacologic study. Neuro-Oncology 7, 177-182 (2005).

2. Baker, S.D. et al. Complete Response of a Recurrent, Multicentric Malignant Glioma in a Patient Treated with Phenylbutyrate. J Neuro-Oncol. 59, 239-242 (2002).

3. Qian, K. et al. Sodium Phenylbutyrate Inhibits Tumor Growth and the Epithelial-Mesenchymal Transition of Oral Squamous Cell Carcinoma In Vitro and In Vivo. Cancer Biother Radiopharm. 33, 155-161 (2018).

4. Ferriero, R. et al. Differential inhibition of PDKs by phenylbutyrate and enhancement of pyruvate dehydrogenase complex activity by combination with dichloroacetate. J Inherit Metab Dis. 38, 895-904 (2015).

5. Goh, M. et al. Phenylbutyrate attenuates the expression of Bcl-XL, DNA-PK, caveolin-1, and VEGF in prostate cancer cells. Neoplasia 3, 331-8 (2001).

⚠️ Important Information: This content is for informational and educational purposes only. It is based on scientific research but is not medical advice. Phenylbutyrate can interact with medications and may not be suitable for everyone. Always consult with a qualified healthcare professional before considering any treatment, particularly for serious conditions like cancer. Phenylbutyrate should never replace conventional cancer treatment unless under the guidance of qualified oncologists.

Last updated: September 2025

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

Niclosamide: Multi-Targeted Cancer Therapeutic with Nanomolar Potency

From Anthelmintic to Anticancer: A Repurposed Drug with Exceptional Blood Cancer Activity

Niclosamide emerges as a remarkably potent anticancer agent with IC50 values ranging from 0.15-5 μM across cancer types, demonstrating exceptional activity against hematological malignancies at nanomolar concentrations. This FDA-approved anthelmintic targets multiple oncogenic pathways simultaneously—Wnt/β-catenin, STAT3, NF-κB—while inducing ferroptosis and enhancing immunotherapy responses. Despite bioavailability challenges, novel formulations achieve therapeutic plasma levels, positioning niclosamide for combination cancer therapy.

Niclosamide

Discovery and Drug Repurposing Journey

Niclosamide represents one of oncology's most compelling drug repurposing success stories. Originally developed in 1958 by Bayer as an anthelmintic for treating tapeworm infections, this salicylanilide derivative gained FDA approval and has been safely used for over six decades. The drug's anticancer potential emerged serendipitously during the 2000s when researchers investigating Wnt pathway inhibitors discovered niclosamide's remarkable ability to disrupt β-catenin signaling—a critical oncogenic driver in numerous cancers.

The transition from antiparasitic to anticancer agent accelerated after 2011, when multiple independent research groups demonstrated niclosamide's broad-spectrum activity against diverse cancer cell lines. Unlike targeted therapies that address single pathways, niclosamide emerged as a multi-targeted agent capable of simultaneously disrupting multiple oncogenic networks. This polypharmacological approach addresses a fundamental challenge in cancer therapy: the tendency of tumors to develop resistance through pathway redundancy and compensation.

Exceptional Anticancer Potency Across Cancer Types

Comprehensive IC50 profiling reveals niclosamide's remarkable potency hierarchy, with hematological malignancies demonstrating extraordinary sensitivity. Acute myeloid leukemia cells show the most dramatic responses, with HL60 cells exhibiting an IC50 of just 0.28 μM. Primary AML patient samples display even greater sensitivity at 0.129 μM median IC50, while leukemic stem cells (CD34+CD38-) respond at an unprecedented 0.0198 μM—representing exceptional selectivity with an 18-36 fold therapeutic window versus normal bone marrow cells.

Cancer Type-Specific IC50 Values:

Acute Myeloid Leukemia: 0.129-0.28 μM (Primary samples and HL60)
Triple-Negative Breast Cancer: 0.153 μM (SUM159 cells)
Colorectal Cancer: 1.0-6.0 μM (HCT116, SW480, DLD1)
Lung Cancer (NSCLC): 1.7-4.3 μM (A549, H460)
Pancreatic Cancer: 5.9-10 μM (PANC-1, MIA PaCa-2)
Normal Colon Epithelial: >20 μM (10-fold selectivity)

Comparative Potency Analysis

Niclosamide demonstrates exceptional potency with IC50 values of 0.15-5 μM, ranking +1 relative to shikonin's reference standard. Blood cancers show the highest sensitivity, particularly AML at nanomolar concentrations.

Niclosamide achieves superior potency compared to most natural compounds, with particular efficacy against hematological malignancies. AML cells (0.129-0.28 μM) show 20-fold greater sensitivity than solid tumors, while maintaining excellent selectivity versus normal cells (>20 μM).

Multi-Targeted Anticancer Mechanisms

Niclosamide's anticancer efficacy stems from its unprecedented ability to simultaneously disrupt multiple oncogenic pathways, creating a synthetic lethal environment for cancer cells while sparing normal tissues. This polypharmacological approach addresses a fundamental weakness of single-target therapies: cancer's ability to develop resistance through pathway compensation and redundancy.

Mechanism	Cancer Type(s)	Key Findings
Wnt/β-Catenin Inhibition	Colorectal, Gastric	Reduces metastasis-associated protein S100A4
STAT3 Inhibition	NSCLC, Various Solid Tumors	Enhances radiosensitivity and T-cell activation
Oxidative Stress and Ferroptosis	Triple-Negative Breast Cancer (TNBC)	Reduces glutathione and induces ferroptosis
Mitochondrial Uncoupling	Multiple Solid Tumors	Impairs cancer cell energy production
CREB-Dependent Pathway Suppression	Acute Myeloid Leukemia (AML)	Induces apoptosis while sparing normal cells
Immune Checkpoint Sensitization	NSCLC	Prolongs survival in combination with immunotherapy

Wnt/β-Catenin Pathway Disruption

Niclosamide's primary mechanism involves comprehensive dismantling of Wnt signaling architecture through degradation of the co-receptor LRP6, preventing β-catenin stabilization and nuclear translocation. The drug also induces autophagy-mediated degradation of Frizzled1 and Dishevelled-2 proteins, creating multiple points of pathway disruption. In colorectal cancer, this results in dramatic downregulation of S100A4, a metastasis-promoting protein, reducing invasive potential and improving outcomes for patients with high Wnt/β-catenin activity.

STAT3 Inhibition and Immune Enhancement

With an IC50 of 0.25 μM for STAT3 activity, niclosamide outperforms many dedicated STAT3 inhibitors. The drug prevents both Y705 and Y727 phosphorylation, blocking STAT3 dimerization and nuclear translocation. This disrupts a critical survival pathway frequently hyperactivated in resistant cancers. In non-small cell lung cancer, STAT3 inhibition reverses radioresistance while simultaneously downregulating PD-L1 expression, enhancing T-cell infiltration and tumor recognition. This dual mechanism creates synergy with immune checkpoint inhibitors.

Ferroptosis Induction in Treatment-Resistant Cancers

Niclosamide induces ferroptosis—an iron-dependent form of programmed cell death—by inhibiting the System Xc- cystine/glutamate antiporter at 0.1-0.2 μM. This depletes cellular glutathione while suppressing GPX4 expression, creating overwhelming oxidative stress. Combined with mitochondrial uncoupling that generates reactive oxygen species, this mechanism proves particularly effective against triple-negative breast cancer cells that rely heavily on antioxidant defenses. The drug's protonophore activity further disrupts cellular energetics, causing ATP depletion and AMPK activation.

Clinical Development and Bioavailability Challenges

The transition from preclinical promise to clinical reality has revealed niclosamide's primary limitation: extremely poor oral bioavailability of just 5-10%. Standard formulations achieve peak plasma concentrations of only 0.25-6.0 μg/mL after 2g doses—often below the 0.2 μM threshold required for anticancer activity. This pharmacokinetic challenge has driven intensive formulation research and strategic clinical trial design.

Clinical Trial Progress: The NIKOLO trial (NCT02519582) in metastatic colorectal cancer using 2g daily oral niclosamide has completed recruitment but results remain pending. The PDMX1001 reformulation achieved therapeutic plasma levels (0.31-0.65 μM) in Phase Ib prostate cancer trials, with 62.5% of patients achieving PSA responses when combined with abiraterone.

Advanced Formulation Strategies

Multiple approaches address niclosamide's solubility limitations. PLGA nanoparticles with MUC1 aptamer targeting achieve 76% apoptosis in breast cancer cells versus 51% for non-targeted formulations. Cyclodextrin inclusion complexes provide 20-fold solubility improvement, while the PDMX1001 acetate prodrug successfully achieved therapeutic plasma concentrations in clinical trials. Amorphous solid dispersions using PEG 6000 enhance dissolution by 4.4-fold, maintaining the drug in a high-energy state that improves absorption.

Safety Profile and Tolerability

Niclosamide's six-decade safety record provides crucial advantages for cancer repurposing. The maximum tolerated dose of 1200mg three times daily represents a 3.6-fold increase over antiparasitic dosing while maintaining acceptable tolerability. Gastrointestinal effects—primarily diarrhea, nausea, and abdominal discomfort—remain predominantly grade 1-2. Importantly, niclosamide lacks organ-specific toxicities that limit many cancer therapeutics, showing no significant hepatotoxicity, nephrotoxicity, cardiotoxicity, or bone marrow suppression.

Synergistic Combinations and Drug Resistance Reversal

Strategic combination approaches amplify niclosamide's efficacy while circumventing resistance mechanisms that limit monotherapy responses. The drug demonstrates true synergy rather than additive effects across multiple cancer types, with combination indices consistently below 1. Most remarkably, niclosamide reverses multidrug resistance by elevating reactive oxygen species to levels that overwhelm P-glycoprotein-mediated drug efflux.

Chemotherapy Sensitization

Niclosamide demonstrates remarkable ability to overcome platinum resistance. In cisplatin-resistant ovarian cancer cells, niclosamide reverses resistance by suppressing lung resistance-related protein and reversing epithelial-mesenchymal transition. The combination reduces required cisplatin doses by 2-4 fold while maintaining efficacy, potentially mitigating platinum-associated toxicities. In erlotinib-resistant lung cancer, niclosamide blocks compensatory STAT3 activation, achieving complete tumor regression in 25% of treated mice versus progressive disease with monotherapy.

Immunotherapy Enhancement

Niclosamide's PD-L1 suppression enhances checkpoint blockade efficacy through both STAT3 inhibition and disruption of HuR-mediated mRNA stabilization. In triple-negative breast cancer models, combining niclosamide with anti-PD-L1 antibodies increased tumor-infiltrating CD8+ T cells by 3-fold and extended median survival from 35 to 62 days. The drug also demonstrates remarkable radiosensitization properties, with enhancement ratios of 1.5-2.3 across cancer types by preventing radiation-induced STAT3 activation and HIF-1α upregulation.

Future Directions and Precision Medicine Applications

The evolving research landscape reveals strategic movement toward biomarker-driven precision approaches rather than broad anticancer applications. Recent mechanistic studies identified phosphorylated JNK as a common regulator of niclosamide-induced autophagy and apoptosis, providing a potential predictive biomarker. S100A4 expression emerges as another selection criterion, particularly for colorectal cancer where this metastasis-associated protein drives disease progression.

Cancer Stem Cell Targeting

Cancer stem cell populations represent an increasingly important target, with niclosamide showing 5-10 fold greater activity against CSCs versus bulk tumor cells. The drug reduces mammosphere formation, ALDH activity, and CD44high/CD24low populations—functional markers of stemness associated with recurrence and metastasis. DCLK1-B expression in colorectal cancer and CAIX expression in triple-negative breast cancer offer additional biomarkers for patient stratification.

Conclusion

Niclosamide exemplifies successful drug repurposing, transforming from a simple anthelmintic to a sophisticated multi-targeted cancer therapeutic. Its exceptional potency against hematological malignancies, combined with unique mechanisms targeting ferroptosis and immune enhancement, creates distinctive therapeutic opportunities. While bioavailability challenges have slowed clinical translation, recent formulation advances and growing mechanistic understanding position niclosamide for expanded development.

Success will likely emerge through precision medicine approaches that match niclosamide's diverse mechanisms with specific tumor vulnerabilities. The convergence of improved delivery systems, validated biomarkers, and rational combinations suggests this 60-year-old drug may finally realize its potential as a cancer therapeutic. For patients with limited options, particularly those with resistant or metastatic disease, niclosamide offers hope that existing medicines can be reimagined to extend and improve lives.

Key Research Citations

1. Burock S, Daum S, Keilholz U, et al. 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. 2018;18(1):297.

2. Jin Y, Lu Z, Ding K, et al. Antineoplastic mechanisms of niclosamide in acute myelogenous leukemia stem cells: inactivation of the NF-kappaB pathway and generation of reactive oxygen species. Cancer Res. 2010;70(6):2516-27.

3. Chae HD, Cox N, Dahl GV, et al. Niclosamide suppresses acute myeloid leukemia cell proliferation through inhibition of CREB-dependent signaling pathways. Oncotarget. 2018;9(3):3301-3317.

4. Mathew M, Sivaprakasam S, Dharmalingam-Nandagopal G, et al. 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. 2024;13(3):291.

5. You S, Li R, Park D, et al. Phase Ib trial of reformulated niclosamide with abiraterone/prednisone in men with castration-resistant prostate cancer. Sci Rep. 2021;11(1):6442.

6. Luo F, Luo M, Rong QX, et al. Niclosamide, an antihelmintic drug, enhances efficacy of PD-1/PD-L1 immune checkpoint blockade in non-small cell lung cancer. J Immunother Cancer. 2019;7(1):245.

⚠️ Important Information: This content is for informational and educational purposes only. It is based on scientific research but is not medical advice. Niclosamide and related compounds can interact with medications and may not be suitable for everyone. Always consult with a qualified healthcare professional before considering any compound for health purposes, particularly for serious conditions like cancer. Experimental compounds should never replace conventional cancer treatment unless under the guidance of qualified oncologists.

Last updated: September 2025