Genistein potentiates the antitumor effect of 5-Fluorouracil by inducing apoptosis and autophagy in human pancreatic cancer cells.
ABSTRACT: Although 5-fluorouracil (5-FU)-based combination chemotherapy (i.e. FOLFIRINOX) has demonstrated effectiveness against pancreatic cancer, novel therapeutic strategies must be developed to increase the therapeutic window of these cytotoxic agents. Genistein is a soy-derived isoflavone with pleiotropic biological effects that can enhance the antitumor effect of chemotherapeutic agents.To understand how genistein potentiates the antitumor effects of 5-FU, we examined apoptosis and autophagy in MIA PaCa-2 human pancreatic cancer cells and their derived xenografts. Apoptosis was evaluated using DNA fragmentation assays, and western blots of poly(ADP ribose)polymerase and caspase-3. Meanwhile, autophagy was evaluated using western blots of microtubule-associated protein light chain 3 (LC3)-I/II, fluorescent microscopy observation of green fluorescent protein-LC3B puncta formation, and acidic vesicular organelle formation using acridine orange staining. Tumors from animal treatment studies were examined for apoptosis and autophagy using the TdT-mediated dUTP nick-end labeling assay and immunohistochemical staining of LC3B, respectively.We observed that genistein increased 5-FU-induced cell death through increased apoptosis, as well as autophagy. The increased autophagy was accompanied by decreased B-cell lymphoma 2 (Bcl2) and increased beclin-1 protein levels. Animal treatment studies supported these observations. The combination of 5-FU and genistein significantly reduced final xenograft tumor volume when compared to 5-FU-alone by inducing apoptosis as well as autophagy.Genistein can potentiate the antitumor effect of 5-FU by inducing apoptotic as well as autophagic cell death. These results demonstrate the potential of genistein as an adjuvant therapeutic agent against pancreatic cancer.
Project description:Modulation of autophagy has been increasingly regarded as a promising cancer therapeutic approach. In this study, we screened several ginsenosides extracted from Panax ginseng and identified ginsenoside Ro (Ro) as a novel autophagy inhibitor. Ro blocked the autophagosome-lysosome fusion process by raising lysosomal pH and attenuating lysosomal cathepsin activity, resulting in the accumulation of the autophagosome marker MAP1LC3B/LC3B and SQSTM1/p62 (sequestosome 1) in various esophageal cancer cell lines. More detailed studies demonstrated that Ro activated ESR2 (estrogen receptor 2), which led to the activation of NCF1/p47(PHOX) (neutrophil cytosolic factor 1), a subunit of NADPH oxidase, and subsequent reactive oxygen species (ROS) production. Treatment with siRNAs or inhibitors of the ESR2-NCF1-ROS axis, such as N-acetyl-L-cysteine (NAC), diphenyleneiodonium chloride (DPI), apocynin (ACN), Tiron, and Fulvestrant apparently decreased Ro-induced LC3B-II, GFP-LC3B puncta, and SQSTM1, indicating that ROS instigates autophagic flux inhibition triggered by Ro. More importantly, suppression of autophagy by Ro sensitized 5-fluorouracil (5-Fu)-induced cell death in chemoresistant esophageal cancer cells. 5-Fu induced prosurvival autophagy, and by inhibiting such autophagy, siRNAs against BECN1/beclin 1, ATG5, ATG7, and LC3B enhanced 5-Fu-induced autophagy-associated and apoptosis-independent cell death. We observed that Ro potentiates 5-Fu cytotoxicity via delaying CHEK1 (checkpoint kinase 1) degradation and downregulating DNA replication process, resulting in the delayed DNA repair and the accumulation of DNA damage. In summary, these data suggest that Ro is a novel autophagy inhibitor and could function as a potent anticancer agent in combination therapy to overcome chemoresistance.
Project description:BACKGROUND:Tubeimoside-I (TBM), a plant-derived bioactive compound, shows antitumor activity in different tumors and can enhance the efficacy of chemotherapeutic agents. However, the detail mechanism underlying remains to be elucidated. METHODS:The cytotoxic potential of TBM towards CRC cells was examined by CCK8 assay, colony formation, LDH release assay, flow cytometry method and Western blots. The ROS levels, autophagy, apoptosis, chemosensitivity to 5-FU or DOX, etc. were determined between control and TBM-treated CRC cells. RESULTS:In this study, we found that TBM could inhibit proliferation and induce apoptosis in colorectal cancer (CRC) cells. Intriguingly, TBM treatment could either promote autophagy initiation by ROS-induced AMPK activation, or block autophagy flux through inhibiting lysosomal hydrolytic enzymes, which leaded to massive impaired autophagylysosomes accumulation. Administration of autophagy initiation inhibitor (3-MA or selective ablation of autophagy related proteins) relieves TBM-induced CRC suppression, while combination use of autophagy flux inhibitor chloroquine (CQ) slightly augments TBM-induced cell death, suggesting that impaired autophagylysosomes accumulation contributes to TBM-induced growth inhibition in CRC cells. Notably, as an autophagy flux inhibitor, TBM works synergistically with 5-fluorouracil (5-FU) or doxorubicin (DOX) in CRC suppression. CONCLUSION:Together, our study provides new insights regarding the anti-tumor activity of TBM against CRC, and established potential applications of TBM for CRC combination therapies in clinic.
Project description:Triple negative breast cancer (TNBC) is very aggressive and lacks specific therapeutic targets, having limited treatment options and poor prognosis. -gingerol is the most abundant and studied compound in ginger, presenting diverse biological properties such as antitumor activity against several types of cancer, including breast cancer. In this study, we show that the semi-synthetic analogue SSi6, generated after chemical modification of the -gingerol molecule, using acetone-2,4-dinitrophenylhydrazone (2,4-DNPH) reagent, enhanced selective cytotoxic effects on MDA-MB-231 cells. Remarkably, unlike the original -gingerol molecule, SSi6 enabled autophagy followed by caspase-independent apoptosis in tumor cells. We found a time-dependent association between SSi6-induced oxidative stress, autophagy and apoptosis. Initial SSi6-induced reactive oxygen species (ROS) accumulation (1h) led to autophagy activation (2-6h), which was followed by caspase-independent apoptosis (14h) in TNBC cells. Additionally, our data showed that SSi6 induction of ROS plays a key role in the promotion of autophagy and apoptosis. In order to investigate whether the observed cell death induction was dependent on preceding autophagy in MDA-MB-231 cells, we used siRNA to knock down LC3B prior to SSi6 treatment. Our data show that LC3B downregulation decreased the number of apoptotic cells after treatment with SSi6, indicating that autophagy is a key initial step on SSi6-induced caspase-independent apoptosis. Overall, the results of this study show that structural modifications of natural compounds can be an interesting strategy for developing antitumor drugs, with distinct mechanisms of actions, which could possibly be used against triple negative breast cancer cells that are resistant to canonical apoptosis-inducing drugs.
Project description:The EI24 autophagy-associated transmembrane protein is frequently associated with tumor growth and patient survival. In the present study, we found that EI24 was downregulated in pancreatic ductal adenocarcinoma (PDAC) tissues compared with adjacent normal tissues and was associated with cancer cell differentiation. Overexpression of EI24 suppressed cancer cell growth <i>in vitro</i> and <i>in vivo</i> and induced cell cycle S phase arrest, with no impact on caspase-dependent apoptosis. EI24 overexpression also resulted in reduced c-Myc expression, an oncogene in PDAC, accompanied with increased LC3B-II formation, increased Beclin-1, and diminished p62. Together, we propose that EI24 suppresses cell proliferation and prompts cell cycle arrest in pancreatic cancer cells by activating the autophagic lysosomal degradation of c-Myc. Our results suggest a potential mechanism underlying the antitumor effects of EI24 in PDAC and provide insight into the crosstalk between autophagy and cell proliferation involving a possible EI24/Beclin-1/p62/c-Myc signaling pathway.
Project description:Background:Antibody drug conjugate (ADC) showed potent therapeutic efficacy in several types of cancers. The role of autophagy in antitumor effects of ADC remains unclear. Methods:In this study, the ADC, Rituximab-monomethyl auristatin E (MMAE) with a Valine-Citrulline cleavable linker, was designed to investigate its therapeutic efficacy against non-Hodgkin lymphoma (NHL) as well as the underlying mechanisms. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) was used to detect growth inhibition in B-cell lymphoma cell lines, Ramos and Daudi cells, which were treated by Rituximab-MMAE alone or combined with autophagy conditioner. Apoptosis was detected by flow cytometry and immunohistochemistry, and apoptosis inhibitor was employed to discover the relationship between autophagy and apoptosis during the Rituximab-MMAE treatment. Autophagy was determined by three standard techniques which included confocal microscope, transmission electron microscope, and western blots. Ramos xenograft tumors in BALB/c nude mice were established to investigate the antitumor effect of combination use of Rituximab-MMAE and autophagy conditioner in B-NHL therapy. Results:Our results showed that Rituximab-MMAE elicited caspase-3-dependent apoptosis in NHL cells and exhibited potent therapeutic efficacy in vivo. Autophagy, which was characterized by upregulated light chain 3-II expression, and accumulation of autophagosomes, was triggered during the Rituximab-MMAE treatment. Meanwhile, inactivation of Akt/mTOR pathway was shown to be involved in the autophagy triggered by Rituximab-MMAE, indicating a probable mechanism of the ADC-initiated autophagy. Importantly, inhibition of autophagy by chloroquine suppressed the Rituximab-MMAE-induced apoptosis, while activating autophagy by rapamycin significantly enhanced the therapeutic effect of Rituximab-MMAE both in vitro and in vivo. Conclusion:Our data elucidated the critical relationship between autophagy and apoptosis in Rituximab-MMAE-based therapy and highlighted the potential approach for NHL therapy by combined administration of the ADC and autophagy activator.
Project description:Cancer cells display abnormal redox metabolism. Autophagy, anoikis and reactive oxygen species (ROS) play a regulatory role during metastasis. LC3 is a well-known essential molecule for autophagy. Therefore, we wanted to explore the molecular interplay between autophagy, anoikis, and ROS in relation to LC3B. We observed enhanced LC3B level along with increased expression of p62 and modulation of other autophagy-related molecules (Atg 3, 5, 7, 12, 16L1 and Beclin1) by inducing oxidative-stress in ovarian cancer cells using a ROS-producing pro-oxidant molecule. Surprisingly, enhanced LC3B was unable to induce autophagosome formation rather promoted anoikis. ROS-induced inhibition of autophagosome-formation is possibly due to the instability of autophagy initiator, ULK1 complex. Moreover, such upregulation of LC3B via ROS enhanced several apoptotic molecules. Silencing LC3B reduced these apoptotic molecules and increased when overexpressed, suggesting its role in apoptosis. Furthermore, LC3B-dependent apoptosis was decreased by inhibiting ROS, indicating a possible link between ROS, LC3B, and apoptosis. Additionally, ROS-induced enhanced LC3B promoted detachment-induced cell death (anoikis). This was further reflected by reduced cell adhesion molecules (integrin-?3 and focal adhesion kinase) and mesenchymal markers (snail and slug). Our in vitro experimental data was further confirmed in primary tumors developed in syngeneic mice, which also showed ROS-mediated LC3B enhancement along with reduced autophagosomes, integrin-?3 and focal adhesion kinase ultimately leading to the decreased tumor mass. Additionally, primary cells from high-grade serous carcinoma patient's ascites exhibited LC3B enhancement and autophagy inhibition through ROS which provided a clinical relevance of our study. Taken together, this is the first evidence for a non-canonical role of LC3B in promoting anoikis in contrast to autophagy and may, therefore, consider as a potential therapeutic target molecule in ovarian cancer. Taken together, autophagy-inhibition may be an alternative approach to induce apoptosis/anoikis in cancer.
Project description:5-Fluorouracil (5-FU)-based combination therapies are standard treatments for gastrointestinal cancer, where the modulation of autophagy is becoming increasingly important in offering effective treatment for patients in clinical practice. This review focuses on the role of autophagy in 5-FU-induced tumor suppression and cancer therapy in the digestive system.All articles published in English from 1996 to date those assess the synergistic effect of autophagy and 5-FU in gastrointestinal cancer therapy were identified through a systematic online search by use of PubMed. The search terms were "autophagy" and "5-FU" and ("colorectal cancer" or "hepatocellular carcinoma" or "pancreatic adenocarcinoma" or "esophageal cancer" or "gallbladder carcinoma" or "gastric cancer").Critical reviews on relevant aspects and original articles reporting in vitro and/or in vivo results regarding the efficiency of autophagy and 5-FU in gastrointestinal cancer therapy were reviewed, analyzed, and summarized. The exclusion criteria for the articles were as follows: (1) new materials (e.g., nanomaterial)-induced autophagy; (2) clinical and experimental studies on diagnostic and/or prognostic biomarkers in digestive system cancers; and (3) immunogenic cell death for anticancer chemotherapy.Most cell and animal experiments showed inhibition of autophagy by either pharmacological approaches or via genetic silencing of autophagy regulatory gene, resulting in a promotion of 5-FU-induced cancer cells death. Meanwhile, autophagy also plays a pro-death role and may mediate cell death in certain cancer cells where apoptosis is defective or difficult to induce. The dual role of autophagy complicates the use of autophagy inhibitor or inducer in cancer chemotherapy and generates inconsistency to an extent in clinic trials.Autophagy might be a therapeutic target that sensitizes the 5-FU treatment in gastrointestinal cancer.
Project description:Pancreatic tumors exhibit enhanced autophagy as compared to any other cancer, making it resistant to chemotherapy. We evaluated the effect of penfluridol against pancreatic cancer. Penfluridol treatment induced apoptosis and inhibited the growth of Panc-1, BxPC-3 and AsPC-1, pancreatic cancer cells with IC50 ranging between 6-7 μM after 24 h of treatment. Significant autophagy was induced by penfluridol treatment in pancreatic cancer cells. Punctate LC3B and autophagosomes staining confirmed autophagy. Inhibiting autophagy by chloroquine, bafilomycin, 3-methyladenine or LC3BsiRNA, significantly blocked penfluridol-induced apoptosis, suggesting that autophagy lead to apoptosis in our model. Penfluridol treatment suppressed the growth of BxPC-3 tumor xenografts by 48% as compared to 17% when treated in combination with chloroquine. Similarly, penfluridol suppressed the growth of AsPC-1 tumors by 40% versus 16% when given in combination with chloroquine. TUNEL staining and caspase-3 cleavage revealed less apoptosis in the tumors from mice treated with penfluridol and chloroquine as compared to penfluridol alone. Penfluridol treatment also suppressed the growth of orthotopically implanted Panc-1 tumors by 80% by inducing autophagy-mediated apoptosis in the tumors. These studies established that penfluridol inhibits pancreatic tumor growth by autophagy-mediated apoptosis. Since penfluridol is already in clinic, positive findings from our study will accelerate its clinical development.
Project description:Ginsenosides have been reported to possess various pharmacological effects, including anticancer effects. Nevertheless, there are few reports about the antitumor activity and mechanisms of ginsenoside Rg5 against breast cancer cells. In the present study, the major ginsenoside Rb1 was transformed into the rare ginsenoside Rg5 through enzymatic bioconversion and successive acid-assisted high temperature and pressure processing. Ginsenosides Rb1, Rg3, and Rg5 were investigated for their antitumor effects against five human cancer cell lines via the MTT assay. Among them, Rg5 exhibited the greatest cytotoxicity against breast cancer. Moreover, Rg5 remarkably suppressed breast cancer cell proliferation through mitochondria-mediated apoptosis and autophagic cell death. LC3B-GFP/Lysotracker and mRFP-EGFP-LC3B were utilized to show that Rg5 induced autophagosome-lysosome fusion. Western blot assays further illustrated that Rg5 decreased the phosphorylation levels of PI3K, Akt, mTOR, and Bad and suppressed the PI3K/Akt signaling pathway in breast cancer. Moreover, Rg5-induced apoptosis and autophagy could be dramatically strengthened by the PI3K/Akt inhibitor LY294002. Finally, a molecular docking study demonstrated that Rg5 could bind to the active pocket of PI3K. Collectively, our results revealed that Rg5 could be a potential therapeutic agent for breast cancer treatment.
Project description:Chronic obstructive pulmonary disease (COPD) is a progressive lung disease characterized by abnormal cellular responses to cigarette smoke, resulting in tissue destruction and airflow limitation. Autophagy is a degradative process involving lysosomal turnover of cellular components, though its role in human diseases remains unclear.Increased autophagy was observed in lung tissue from COPD patients, as indicated by electron microscopic analysis, as well as by increased activation of autophagic proteins (microtubule-associated protein-1 light chain-3B, LC3B, Atg4, Atg5/12, Atg7). Cigarette smoke extract (CSE) is an established model for studying the effects of cigarette smoke exposure in vitro. In human pulmonary epithelial cells, exposure to CSE or histone deacetylase (HDAC) inhibitor rapidly induced autophagy. CSE decreased HDAC activity, resulting in increased binding of early growth response-1 (Egr-1) and E2F factors to the autophagy gene LC3B promoter, and increased LC3B expression. Knockdown of E2F-4 or Egr-1 inhibited CSE-induced LC3B expression. Knockdown of Egr-1 also inhibited the expression of Atg4B, a critical factor for LC3B conversion. Inhibition of autophagy by LC3B-knockdown protected epithelial cells from CSE-induced apoptosis. Egr-1(-/-) mice, which displayed basal airspace enlargement, resisted cigarette-smoke induced autophagy, apoptosis, and emphysema.We demonstrate a critical role for Egr-1 in promoting autophagy and apoptosis in response to cigarette smoke exposure in vitro and in vivo. The induction of autophagy at early stages of COPD progression suggests novel therapeutic targets for the treatment of cigarette smoke induced lung injury.