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Three platinum(II) complexes of dicyclopentadiene (DCP) and dithiocarbamates (DTCs), namely, [Pt(η4-DCP)(Me2DTC)]PF6 (1), [Pt(η4-DCP)(Et2DTC)]PF6 (2), and [Pt(η4-DCP)(Bz2DTC)]PF6 (3) [Me2DTC = dimethyldithiocarbamate, Et2DTC = diethyldithiocarbamate, and Bz2DTC = dibenzyldithiocarbamate] were prepared and characterized by elemental analysis, IR, 1H, and 13C Nuclear Magnetic Resonance spectroscopy. The spectroscopic data indicated the coordination of both DCP and DTC ligands to platinum(II). The solution chemistry of complex 1 revealed that the complexes are stable in both dimethyl sulfoxide (DMSO) and 1:1 mixture of DMSO:H2O. In vitro cytotoxicity of the complexes relative to cisplatin was tested using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, against CHL-1 (human melanoma cancer cells), MDA-MB-231 (breast cancer cells), A549 (lung cancer cells), and B16 (murine melanoma cancer cells). The antiproliferative effect of all three prepared complexes was found to be significantly higher than cisplatin. Furthermore, flow cytometric analysis of complex 1 showed that the complex induced apoptosis, oxidative stress, mitochondrial potential depolarization and cell cycle arrest in a concentration-dependent pattern in the CHL-1 cells. Confirmation of apoptosis via gene expression analysis demonstrated down-regulation of anti-apoptotic genes and up-regulation of pro-apoptotic genes in the CHL-1 cells. Wound-healing assays also lent support to the strong cytotoxicity of the complexes. In vivo studies showed a significant reduction of tumor volume at the end of the experiment. In addition, the drug did not change the weight of the mice. In conclusion, complex 1 inhibited cell proliferation in vitro and reduced tumor growth in vivo.

Tumor cells undergo senescence in response to both conventional and targeted cancer therapies. The induction of senescence in response to cancer therapy can contribute to unfavorable patient outcomes, potentially including disease relapse. This possibiliy is supported by our findings that tumor cells induced into senescence by doxorubicin or etoposide can give rise to viable tumors in vivo. We further demonstrate sensitivity of these senescent tumor cells to the senolytic ABT‐263 (navitoclax), therefore providing a “two‐hit” approach to eliminate senescent tumor cells that persist after exposure to chemotherapy or radiation. The sequential combination of therapy‐induced senescence and ABT‐263 could shift the response to therapy toward apoptosis by interfering with the interaction between BCL‐XL and BAX. The administration of ABT‐263 after either etoposide or doxorubicin also resulted in marked, prolonged tumor suppression in tumor‐bearing animals. These findings support the premise that senolytic therapy following conventional cancer therapy may improve therapeutic outcomes and delay disease recurrence.

Gefitinib (GEF) is a selective inhibitor of the epidermal growth factor receptor (EGFR) used to treat non-small cell lung cancer. Yet, few cases of cardiotoxicity have been reported. However, the role of the PTEN/Akt/FoxO3a pathway, which mediates GEF anticancer activity, in GEF cardiotoxicity remains unclear. For this purpose, in vitro H9c2 cells and in vivo rat cardiomyocytes were utilized as study models. Treatment of H9c2 cells and Sprague-Dawley rats with GEF significantly induced the expression of hypertrophic and apoptotic markers at mRNA and protein levels with an increased plasma level of troponin. This was accompanied by induction of autophagy and mitochondrial dysfunction in H9c2 cells. Inhibition of cardiac EGFR activity and Akt cellular content of in vitro and in vivo rat cardiomyocytes by GEF increased PTEN and FoxO3a gene expression and cellular content. Importantly, treatment of H9c2 cells with PI3K/Akt inhibitor increased PTEN and FoxO3a mRNA expression associated with potentiation of GEF cardiotoxicity. In addition, by using LC-MS/MS, we showed that GEF is metabolized in the rat heart microsomes into one cyanide- and two methoxylamine-adduct reactive metabolites, where their formation was entirely blocked by CYP1A1 inhibitor, α-naphthoflavone. The current study concludes that GEF induces cardiotoxicity through modulating the expression and function of the cardiac PTEN/AKT/FoxO3a pathway and the formation of CYP1A1-mediated reactive metabolites.

There are many mechanisms of resistance, chemoresistance of HeLa cells to anti-cancer agents seems to be autophagy-mediated. While using very effective anti-cancers such as Doxorubicin and cisplatin, cells overcome the cytotoxicity of these drugs through promotion of what so-called cytoprotective autophagy. Here in this study, we sought to introduce a novel platinum-based compound PBC-II that possesses anti-cancer activity. Our data showed that PBC-II is able to induce apoptosis at relatively low concentrations, with no detectable reactive oxygen species (ROS). However, further experiments demonstrated that exposure of HeLa cells to PBC-II did not promote autophagy; rather, it resulted in accumulation of p62 and decrease in LC3-II levels. Autophagy was then promoted in HeLa cells pharmacologically by Doxorubicin and genetically by siRNA IL-10. In order to confirm promotion of autophagy in our model, we performed acridine orange staining to assess for autophagy under microscope as well as via flow cytometry. We then measured protein level of autophagy markers p62 and LC3 by western blot. Our data indicated that PBC-II interferes with therapy-induced autophagy. We also determined PI3K activity while co-incubation of PBC-II with autophagy inducers. It was clear that PI3K activation decreased when PBC-II was co-administered with autophagy inducers. Collectively, PBC-II exerts unique anti-proliferative effects associated with inhibition of autophagy, which indicates that PBC-II is potentially a promising agent to be used in resistant ovarian tumors. Keywords: Ovarian, Autophagy, Doxorubicin, IL-10, Platinum, HeLa

The loss of patentability of many originator biologics has led to the rapid introduction of biosimilar agents. The anticipated economic benefit of introducing such agent has been accompanied by vagueness surrounding their biotechnology, approval requirements, positioning in treatment paradigms and potential for adverse events. The Second Symposium on Biologics and Biosimilars “Beyond Clinical Practice” was held on 24th-26th January 2020 aiming at improving the understanding of these new agents in a diverse interactive conference and to guide stakeholders how to introduce biosimilars into clinical practice. The symposium consisted of 4 tracks and 3 workshops. A total of 217 participants attended the meeting. The majority were pharmacists (78.8%) followed by physicians (18.9%) and other healthcare providers (2.3%). The workshops covered the following topics: basics of pharmacoeconomics, pharmacovigilance and patients’ perspective toward biosimilar biologics. While, the 4 main tracks included: Introduction to biosimilars, challenges in clinical practice, regulatory and pharmacoeconomic aspects and Challenges in biosimilar pharmacovigilance.

Three novel gold(III) complexes (1–3) of general composition [Au(Bipydc)(S2CNR2)]Cl2 (Bipydc = 2,2′-bipyridine-3,3′-dicarboxylic acid and R = methyl for dimethyldithiocarbamate (DMDTC), ethyl for diethyldithiocarbamate (DEDTC), and benzyl for dibenzyldithiocarbamate (DBDTC)) have been synthesized and characterized by elemental analysis, FTIR and NMR spectroscopic techniques. The spectral results confirmed the presence of both the Bipydc and dithiocarbamate ligands in the complexes. The in vitro cytotoxic studies demonstrated that compounds 1–3 were highly cytotoxic to A549, HeLa, MDA-231, and MCF-7 cancer cells with activities much higher (about 25-fold) than cisplatin. In order to know the possible mode of cell death complex 2, [Au(Bipydc)(DEDTC)]Cl2 was further tested for induction of apoptosis towards the MCF-7 cells. The results indicated that complex 2 induces cell death through apoptosis.

Objective: Diabetic complications involve multiple pathological pathways, including hyperglycemia-induced oxidative stress and inflammation. Combination therapy is usually employed to improve treatment outcomes and to lower potential adverse effects. In this study, we evaluated the effects of antidiabetic and antihypertensive agents, glibenclamide (GLI) and losartan (LT), on diabetes mellitus (DM)-associated metabolic changes in rats. Materials and Methods: Streptozotocin-induced diabetic animals were orally treated with GLI 5 mg/kg and/or LT 25 mg/kg for 4 weeks. Blood glucose, insulin, aspartate aminotransferase, alanine aminotransferase, urinary creatinine, and urea levels were measured. Serum, liver, and kidney values of inflammatory markers, such as interleukin-1β, tumor necrosis factor alpha, and interleukin-6 were assessed, along with lipid peroxidation products (e.g., thiobarbituric acid reactive substances), endogenous antioxidants (e.g., glutathione), as well as antioxidant enzyme activities (e.g., catalase, superoxide dismutase, and glutathione peroxidase). Finally, histological changes in liver and kidney tissues were evaluated. Results: DM markedly induced systemic, hepatic, and renal inflammation and lowered antioxidant defense mechanisms. Treatment of diabetic rats with either GLI or LT significantly improved liver and kidney functions and histological structure. Moreover, both medications reduced signs of oxidative stress and inflammation in blood, liver, and kidney samples. Combining GLI and LT showed similar protective potential against systemic, hepatic, and renal oxidative stress and inflammation. Conclusion: Adding LT to GLI therapy revealed prospective antioxidant and anti-inflammatory action, while no synergistic or additive effects were observed.

Metformin (MET) is a clinically used anti-hyperglycemic agent that shows activities against chemically-induced animal models of cancer. A study from our laboratory showed that MET protectes against 7, 12-dimethylbenz[a]anthracene (DMBA)-induced carcinogenesis in vitro human non-cancerous epithelial breast cells (MCF10A) via activation of the aryl hydrocarbon receptor (AhR). However, it is unclear whether MET can prevent the initiation of breast carcinogenesis in an in vivo rat model of AhR-induced breast carcinogenesis. Therefore, the main aims of this study are to examine the effect of MET on protecting against rat breast carcinogenesis induced by DMBA and to explore whether this effect is medicated through the AhR pathway. In this study, treatment of female rats with DMBA initiated breast carcinogenesis though inhibiting apoptosis and tumor suppressor genes while inducing oxidative DNA damage and cell cycle proliferative markers. This effect was associated with activation of AhR and its downstream target genes; cytochrome P4501A1 (CYP1A1) and CYP1B1. Importantly, MET treatment protected against DMBA-induced breast carcinogenesis by restoring DMBA effects on apoptosis, tumor suppressor genes, DNA damage, and cell proliferation. Mechanistically using in vitro human breast cancer MCF-7 cells, MET inhibited breast cancer stem cells spheroids formation and development by DMBA, which was accompanied by a proportional inhibition in CYP1A1 gene expression. In conclusion, the study reports evidence that MET is an effective chemopreventive therapy for breast cancer by inhibiting the activation of CYP1A1/CYP1B1 pathway in vivo rat model.

Autophagy and senescence, predominant responses that may dictate cell fate after chemotherapy or radiation, often occur in tandem. Cells in states of senescence and/or autophagy are frequently growth arrested. We have previously reported that tumor cells induced into senescence by therapy can re-emerge from the growth-arrested state, a phenomenon termed proliferative recovery. The current work shows that, while tumor cells collaterally induced into senescence and autophagy by etoposide, doxorubicin, or radiation undergo proliferative recovery, neither pharmacological nor genetic inhibition of early autophagy alter the extent of senescence or the ability of cells to recover from senescence. These findings confirm and extend our previous observations, essentially dissociating senescence from autophagy, and further indicate that re-emergence from senescence does not appear to be facilitated by or dependent on autophagy. Our results also provide additional evidence for the promotion of the non-protective form of autophagy by both chemotherapeutic drugs and radiation, which may complicate current efforts to inhibit autophagy for therapeutic benefit.

The targeting of pro-inflammatory enzymes becomes a therapeutic intervention when acute inflammation is proliferating in pathological conditions. This research is intended to carry out an evaluation of inhibiting and inducing enzymes with inflammatory associations with 28 cyclohexanone analogs based on the ligustrazine. Tests were undertaken with inhibitor screening assay kits using a range of synthetic compounds to investigate how they could inhibit the activity of cyclooxygenase (COX) enzymes, secretory phospholipase A2 (sPLA2), and lipoxygenase (LOX) enzyme. Significant and similar inhibitory activities against sPLA2 with were noted with synthetic compounds which included 1f and 1g (IC50 = 2.2 μM). The optimal inhibitory activity regarding LOX enzyme was shown with compounds 1d (IC50 = 8.1 μM) and 1e (IC50 = 7.5 μM). Additionally, the compounds 1b, 1d, 1e, 2n, and 2o were shown to be significant inhibitors of COX-1 activity with IC50 values 0.09 to 0.7 μM. The outcomes of assays for COX inhibition demonstrated that the same compounds had a further strong inhibitive influence on the COX-2 enzyme, and certain compounds such as 1d, 1e, and 2n demonstrated enhanced potency compared with positive controls.

Colorectal cancer (CRC) is type of cancer that growing in the colon or the rectum. Although CRC can be treated and managed through several means of therapy, the disease mortality is still high in Saudi Arabia. Upon treatment, cancer cells undergo different form of cell death or growth arrest such as apoptosis and senescence. Senescence is a state of proliferation arrest that is considered as a cause of drug resistance. The current study was designed to interfere with Doxorubicin-induced senescence in cancer cells, and shift the cytotoxicity of doxorubicin from proliferation arrest to apoptosis. β-galactosidase (β-gal) staining has demonstrated that Doxorubicin induces senescence in wild-type HCT116 but not in p21 knock-out cells. (DAPI)/TUNEL stating and Annexin V assay indicate that treatment with Doxorubicin promotes apoptosis in p21 knock-out HCT116 significantly more than in wild-type HCT116. β-galactosidase staining and 5-Dodecanoylaminofluorescein Di-β-D-Galactopyranosid (C12FDG) demonstrated that combination of Doxorubicin with Sorafenib, Rapamycin, or Venetoclax has significantly reduced senescence and induced more apoptosis as shown by Propidium iodide (PI)/Annexin v assay and Caspase 3/7 Assay. PCR data analysis demonstrated that not all combination of Doxorubicin with other agents significantly decreased expression of senescence protein, however, the results of this study provide findings that interference with senescence pathways may shift cytotoxicity from senescence to apoptosis. The authors extend their appreciation to the Deputyship for Research and Innovation, Ministry of Education in Saudi Arabia for partly funding this work through the project number (DRI-KSU-1273). Citation Format: Moureq Rashed Alotaibi, Homood As Sobeia, Munirah Alohaydib, Faten Alaqil, Khalid Alhazzani, Ali Alhoshani, Tareq Saleh, David Gewirtz. Studying the effects of interfering with doxorubicin-induced senescence in human colon cancer HCT116 cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2024.

Background: Acute lymphoblastic leukemia (ALL) is the most common type of childhood malignancy. Despite all the improvements in ALL treatment, cancer relapse remains a major dilemma. Immune checkpoint inhibitors are promising agents that are being evaluated in ALL clinical trials. The overall aim of our study is to evaluate the expression status of immune checkpoint genes in pediatric ALL patients as prognostic factors for the time of relapse and identify possible targets for immunotherapy. Methods: The expression of 21 regulatory immune checkpoint genes was extracted from two microarray datasets of precursor-B-ALL patients (Hogan et al. dataset (GSE28460) included 49 patients and Staal et al. dataset (GSE18497) contained 27 patients). The microarray data were run using the robust multichip average normalization method in RStudio (1.3.959). A 36 month-cutoff was determined to stratify patients into two groups, early relapse (< 36 months) and late relapse (≥ 36 months). The two datasets were normalized to a reference group (late relapse), then combined. The significant differentially expressed genes between the two groups were detected using Student's t-test. An expression score was generated for each patient based on the expression of the significant differentially expressed genes. Survival analysis and Pearson's correlation were performed to assess the impact of significant genes on the time of relapse. Results: 19 out of the 21 genes were downregulated in early relapsing patients compared to the late relapsing group, seven of which were statistically significant; BTLA4, KIR3DS, PDCD1, TIGIT, HAVCR, CTLA4, TNFRSF9. The expression of those seven genes was significantly associated with the time of relapse ( r = 0.381, P = 0.0007). Patients with a low expression profile of the significant differentially expressed genes were 2.3 times more likely to relapse early (CI 1.434-3.713, P = < 0.0001). Conclusion: The expression of immune checkpoint genes is significantly associated with the prognosis of ALL patients. BTLA4, KIR3DS, PDCD1, TIGIT, HAVCR, CTLA4, and TNFRSF9 might be prognostic factors for the time of relapse. Immunotherapy might be beneficial to the late relapsing patients where immune checkpoint genes were upregulated. The study was funded by the deputyship for Research and Innovation, Ministry of Education, Saudi Arabia (DRI-KSU-1273). Citation Format: Basil Jamal Alotaibi, Razan Saad Alshahrani, Munirah Abdulaziz Alkathiri, Ali Mohammed Alqasem, Basil Nasser Alamri, Ali Rashed Alhoshani, Moureq Rashed Alotaibi, Khalid Ahmed Alhazzani, Homood Moqbel As Sobeai. Low expression of immune checkpoint genes is significantly associated with early relapse in pediatric acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3031.

Asthma, a chronic inflammatory disease affecting the airways is primarily caused due to immune system dysfunction. Different inhaled allergens such as house dust mites (HDM), fungi, cockroach allergens are the main contributors to allergic asthma. Protease activated receptor-2 (PAR-2) signaling plays an important role in allergic asthma through modulation of immune mediators in airway epithelial cells (AECs). Interleukin-17A (IL-17A) signals via subunits of IL-17 receptor (IL-17R), i.e. interleukin-17 receptor A (IL-17RA) and interleukin-17 receptor C (IL-17RC), and plays a necessary role in neutrophilic infiltration in response to infectious/allergenic stimuli, however it is not known if PAR-2 activation affects IL-17A/IL-17R signaling during acute exposure to house dust mite (HDM) allergens. Therefore, our study exposed mice to HDM allergens for five days and evaluated its effect on IL-17A/IL-17R signaling, chemokine/cytokines and neutrophilic inflammation in mice. Our study shows that HDM allergens upregulate IL-17A levels in the lung and IL-17RA/IL-17RC expression in AECs. PAR-2 activation by trypsin also upregulates neutrophilic influx and IL-17A/IL-17R signaling in the lung. Upregulated IL-17A/IL-17R signaling was associated with increased BAL neutrophils, pulmonary MPO activity and proinflammatory chemokines and cytokines (IL-23, IL-6, and MCP-1 in AECs/lung) in HDM exposed mice. Further, HDM-induced IL-17A, IL-17R and chemokines/cytokines were attenuated by PAR-2 antagonist, ENMD-1068. Furthermore, HDM-primed mice treated with IL-17A had greater neutrophilic inflammation and higher levels of inflammatory cytokines/chemokines than PBS-exposed mice treated with IL-17A. This proposes that acute exposure to HDM allergens activate AECs at a very early stage where PAR-2/IL-17R signaling serves a crucial role in neutrophilic inflammation.

Studies of radiation interaction with tumor cells often focus on apoptosis as an end point; however, clinically relevant doses of radiation also promote autophagy and senescence. Moreover, functional p53 has frequently been implicated in contributing to radiation sensitivity through the facilitation of apoptosis. To address the involvement of apoptosis, autophagy, senescence and p53 status in the response to radiation, the current studies utilized isogenic H460 non-small cell lung cancer cells that were either p53-wild type (H460wt) or null (H460crp53). As anticipated, radiosensitivity was higher in the H460wt cells than in the H460crp53 cell line; however, this differential radiation sensitivity did not appear to be a consequence of apoptosis. Furthermore, radiosensitivity did not appear to be reduced in association with the promotion of autophagy, as autophagy was markedly higher in the H460wt cells. Despite radiosensitization by chloroquine in the H460wt cells, the radiation-induced autophagy proved to be essentially nonprotective, as inhibition of autophagy via 3-methyl adenine (3-MA), bafilomycin A1 or ATG5 silencing failed to alter radiation sensitivity or promote apoptosis in either the H460wt or H460crp53 cells. Radiosensitivity appeared to be most closely associated with senescence, which occurred earlier and to a greater extent in the H460wt cells. This finding is consistent with the in-depth proteomics analysis on the secretomes from the H460wt and H460crp53 cells (with or without radiation exposure) that showed no significant association with radioresistance-related proteins, whereas several senescence-associated secretory phenotype (SASP) factors were upregulated in H460wt cells relative to H460crp53 cells. Taken together, these findings indicate that senescence, rather than apoptosis, plays a central role in determination of radiosensitivity; furthermore, autophagy is likely to have minimal influence on radiosensitivity under conditions where autophagy takes the nonprotective form.

Background: Breast cancer is affected by the immune system in that different cytokines play roles in its initiation and progression. Interleukin-10 (IL-10), an anti-inflammatory cytokine, is an immunosuppressive factor involved in tumorigenesis. The present study was conducted to investigate the gene silencing effect of a small interference RNA (siRNA) targeting IL-10 on the apoptotic pathway in breast cancer cell line. Methods: The siRNA targeting IL-10 and a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) clone were introduced into MDA-MB-231 cells. Real-time PCR assays were used to determine IL-10 and GAPDH gene expression levels, in addition to those for protein kinase B (AKT), phosphoinositide 3-kinase (PI3K), B-cell lymphoma 2 (Bcl2), caspase-3 and caspase-9 genes related to apoptosis. Results: Inhibition of IL-10 by the siRNA accelerated apoptosis and was accompanied by significant increase in caspase-3 and caspase-9 and a significant decrease in PI3K, AKT and Bcl2 expression levels compared to the non-transfected case. Conclusions: In conclusion, the production of IL-10 may represent a new escape mechanism by breast cancer cells to evade destruction by the immune system. IL-10 gene silencing causes down regulation of both PI3K/AKT and Bcl2 gene expression and also increases the Bbc3, BAX caspase3, and caspase 3 cleavage expression levels. IL–10 might represent a promising new target for therapeutic strategies.

Background Cisplatin is widely used chemotherapeutic agent for cancer treatment with limited uses due to its neurotoxic side effect. The aim of this study was to determine the potential preventive effects of rutin on the brain of cisplatin- neurotoxic rat model. Methods Forty rats were divided into four groups. Group-1 (control group) was intra-peritoneal (IP) injected with 2.5 ml/kg saline. Group-2 (rutin group) was orally administrated 30 mg/kg rutin dissolved in water for 14 days. Group-3 (cisplatin group) was IP received 5 mg/kg cisplatin single dose. Group-4 (rutin and cisplatin group) was orally administrated 30 mg/kg rutin dissolved in water for 14 days with a single dose of 5 mg/kg cisplatin IP on day ten. Brain tissues from frontal cortex was used to extract RNA, the gene expression levels of paraoxonase-1 (PON-1), PON-2, PON-3, peroxisome proliferator-activated receptor delta (PPAR-δ), and glutathione peroxidase (GPx) was investigated by Real-time PCR. Results Cisplatin significantly decreased the expression levels of PON-1, PON-3, PPAR-δ and GPX whereas significantly increased PON-2 expression levels. Co-administration of Rutin prevented the cisplatin-induced toxicity by restoring the alteration in the studied genes to normal values as in the control group. Conclusion This study showed that Rutin has neuroprotective effect and reduces cisplatin- neurotoxicity with possible mechanism via the antioxidant pathway.

Background Cisplatin (CP) is commonly used in the treatment of different types of cancer but nephrotoxicity has been a major limiting factor. Therefore, the present study aimed to study the possible protective effect of rutin against nephrotoxicity induced by cisplatin in rats. Methods Forty male Wistar albino rats were randomly divided into 4 groups. Rats of group 1 control group intraperitoneal (i.p.) received 2.5 ml/kg, group 2 CP group received single dose 5 mg/kg cisplatin i.p. group 3 rutin group orally received 30 mg/kg rutin group 4 (CP plus rutin) received CP and rutin as in group 2 and 3. Kidneys were harvested for histopathology and for the study the gene expression of c-Jun N-terminal kinases (JNK), Mitogen-activated protein kinase 4 (MKK4), MKK7, P38 mitogen-activated protein kinases (P38), tumor necrosis factors alpha (TNF-α), TNF Receptor-Associated Factor 2 (TRAF2), and interleukin-1 alpha (IL-1-α). Results The cisplatin single dose administration to rats induced nephrotoxicity associated with a significant increase in blood urea nitrogen (BUN) and serum creatinine and significantly increase Malondialdehyde (MDA) in kidney tissues by 230 ± 5.5 nmol/g compared to control group. The animal treated with cisplatin showed a significant increase in the expression levels of the IL-1α (260%), TRFA2 (491%), P38 (410%), MKK4 (263%), MKK7 (412%), JNK (680%) and TNF-α (300%) genes compared to control group. Additionally, histopathological examination showed that cisplatin-induced interstitial congestion, focal mononuclear cell inflammatory, cell infiltrate, acute tubular injury with reactive atypia and apoptotic cells. Rutin administration attenuated cisplatin-induced alteration in gene expression and structural and functional changes in the kidney. Additionally, histopathological examination of kidney tissues confirmed gene expression data. Conclusion The present study suggested that the anti-oxidant and anti-inflammatory effect of rutin may prevent CP-induced nephrotoxicity via decreasing the oxidative stress, inhibiting the interconnected ROS/JNK/TNF/P38 MAPK signaling pathways, and repairing the histopathological changes against cisplatin administration.

Autophagy is a process of cellular self-digestion, whereby the cell degrades subcellular materials in order to generate energy and metabolic precursors in order to prolong survival, classically under conditions of nutrient deprivation. Autophagy can also involve the degradation of damaged or aged organelles, and misfolded or damaged proteins to eliminate these components that might otherwise be deleterious to cellular survival. Consequently, autophagy has generally been considered a prosurvival response. Many, if not most chemotherapeutic drugs and radiation also promote autophagy, which is generally considered a cytoprotective response, in that its inhibition frequently promotes apoptotic cells death. Furthermore, it has been shown that conventional chemotherapeutic drugs and radiation alone rarely induce a form of autophagy that leads to cell death. However, there are multiple examples in the literature where newer chemotherapeutic agents, drug combinations or drugs in combination with radiation promote autophagic cell death. This review will describe autophagic cell death induced in breast tumor cells, lung cancer cells as well as glioblastoma, demonstrating that it cannot be concluded that stress induced autophagy is, of necessity, cytoprotective in function.

Senescence represents a fundamental response to cancer therapy. Accumulating senescent cells contribute to the deleterious outcomes of cancer therapy including cancer relapse, effects that may be largely mediated by the Senescence-Associated Secretory Phenotype (SASP). In this work, we show that tumor cells induced into senescence by etoposide retain proliferative capacity based on their capacity to generate proliferating colonies in culture as well as giving rise to viable tumors in vivo. Using a flow cytometry-based enrichment approach based on enlarged size and expression of Senescence-Associated β-galactosidase (SA-β-gal), we were able to utilize real time imaging to establish the re-emergence of non-small cell lung cancer cells from senescence-based arrest and the generation or proliferating daughter cells (i.e. self-renewal). Moreover, we implemented High-Speed Live-Cell Interferometry (HSLCI) to provide a single-cell lineage tracking of dividing senescent cells. The recovery from senescence was accompanied by resolution of several senescence-associated hallmarks, specifically SA-β-gal activity, p21Waf1/Cip1 and several components of the SASP (IL-1β, IL-6 and CXCL1). Our data suggests that Therapy-Induced Senescence (TIS) may ultimately be a transient process in that at least a subpopulation of tumor cells can recover proliferative capacity. We further demonstrate that the senolytic agent, ABT263, which has been shown to eliminate senescent cells from aging-related animal models can also eliminate senescent tumor cells that persistent after exposure to chemotherapy by shifting the response towards apoptotic cell death. Furthermore, sequential administration of ABT263 interferes with the ability of tumor cells induced into senescence by chemotherapy to recover growth potential. These studies suggest that senescent tumor cells can potentially contribute to cancer relapse by acquiring proliferative properties. The use of senolytic agents after induction of senescence by conventional or targeted therapies allows for the clearance of residual (possibly dormant) senescent tumor cells, which could serve to suppress disease recurrence and cancer mortality. Citation Format: Tareq Saleh, Liliya Tyutyunyk-Massey, Graeme F. Murray, Moureq R. Alotaibi, Ajinkya S. Kawale, Zeinab Elsayed, Scott C. Henderson, Vasily Yakovlev, Lynne W. Elmore, Amir Toor, Hisashi Harada, Jason Reed, Joseph W. Landry, David A. Gewirtz. Elimination of senescent tumor cells by ABT263 interferes with proliferative recovery and provides a two-hit therapeutic approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 901.

Radiotherapy continues to be a primary modality in the treatment of cancer. DNA damage induced by radiation can promote apoptosis as well as both autophagy and senescence, where autophagy and senescence can theoretically function to prolong tumor survival. A primary aim of this work was to investigate the hypothesis that autophagy and/or senescence could be permissive for DNA repair, thereby facilitating tumor cell recovery from radiation-induced growth arrest and/or cell death. In addition, studies were designed to elucidate the involvement of autophagy and senescence in radiation sensitization by PARP inhibitors and the re-emergence of a proliferating tumor cell population. In the context of this work, the relationship between radiation-induced autophagy and senescence was also determined. Studies were performed using DNA repair proficient HCT116 colon carcinoma cells and a repair deficient Ligase IV (−/−) isogenic cell line. Irradiation promoted a parallel induction of autophagy and senescence that was strongly correlated with the extent of persistent H2AX phosphorylation in both cell lines; however inhibition of autophagy failed to suppress senescence, indicating that the two responses were dissociable. Irradiation resulted in a transient arrest in the HCT116 cells while arrest was prolonged in the Ligase IV (−/−) cells; however, both cell lines ultimately recovered proliferative function, which may reflect maintenance of DNA repair capacity. The PARP inhibitors (Olaparib) and (Niraparib) increased the extent of persistent DNA damage induced by radiation as well as the extent of both autophagy and senescence; neither cell line underwent significant apoptosis by radiation alone or in the presence of the PARP inhibitors. Inhibition of autophagy failed to attenuate radiation sensitization, indicating that autophagy was not involved in the action of the PARP inhibitors. As with radiation alone, despite sensitization by PARP inhibition, proliferative recovery was evident within a period of 10–20 days. While inhibition of DNA repair via PARP inhibition may initially sensitize tumor cells to radiation via the promotion of senescence, this strategy does not appear to interfere with proliferative recovery, which could ultimately contribute to disease recurrence.

Radiation remains a predominant treatment option for advanced, inoperable lung cancer, however, resistance represents a major barrier against effective therapy. The exact mechanisms through which cancer cells exhibit resistance to radiotherapy still remains unresolved. Traditionally, radiation-induced autophagy is considered as a mechanism of resistance which attenuates the antitumor effects of radiotherapy i.e., autophagy plays a cytoprotective role on cell survival. To examine this concept further, we employed H460 non-small cell lung cancer (NSCLC) cells that were either p53 wild-type (p53 +/+) or p53 null (p53 -/-) to study the contribution of autophagy to radiation sensitivity. While autophagy was markedly higher in p53 +/+ cells (determined by p62 degradation, LC3B conversion, and acidic vesicle formation) the p53 +/+ cells were actually more radiosensitive than their counterpart H460 p53 -/- cells, which showed lower levels of radiation-induced autophagy. Despite modest radiosensitization by chloroquine, alternative pharmacological (3-methly adenine) and genetic (ATG5 silencing) inhibition of autophagy failed to radiosensitize either p53 +/+ or p53 -/- cells, indicating that radiation induced autophagy is not cytoprotective in function in these experimental models. Furthermore, secretome analysis (nLC-MS/MS) of p53 +/+ H460 cells showed no significant association with radioresistance-related proteins in comparison with p53 -/- H460 cells. The rate and extent of apoptosis was quite low and similar in the two cells lines, essentially ruling out apoptotic cell death as the basis for differential radiation sensitivity. Finally, Senescence was more pronounced in the p53 +/+ cells compared to the p53 -/- cells, which may contribute to the greater radiation sensitivity in the p53 +/+ cells. However, the most relevant finding in this work was that when autophagy is the nonprotective form, it does not confer inherent radiation resistance in tumor cells. Citation Format: Jingwen Xu, Emmanuel Kenneth Cudjoe, Tareq Saleh, Nipa Patel, Moureq Alotaibi, Yingliang Wu, Santiago Lima, David Gewirtz. Nonprotective autophagy fails to confer resistance to radiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1326.

While cellular senescence has long been recognized as an irreversible form of growth arrest, evidence in the literature has suggested that subpopulations of senescent tumor cells may retain proliferative capacity. To directly address this question, H460 non-small cell lung cancer cells induced into senescence by exposure to etoposide, and enriched based on β-galactosidase staining and size, were shown to recover reproductive capacity, which was accompanied by resolution of the DNA-damage-response (downregulation of p53 and p21Waf1/Cip1 induction), attenuation of the senescence-associated secretory phenotype (SASP) as well as downregulation of miRNA34 expression and an increase in c-myc. The senescence-enriched lung cancer cells were also capable of tumor growth upon transplantation into immunodeficient mice. Re-emergence from etoposide-induced senescence was also observed using HCT116 colon cancer cells, as evidenced by concomitant downregulation of a senescence-associated reporter (BTG1-RFP). Collectively, these findings indicate that therapy-induced senescence (TIS) may ultimately be a transient process in that at least a subpopulation of tumor cells can resume proliferation. We propose that some forms of tumor dormancy that lead to disease recurrence may reflect cells that enter into and ultimately escape from senescence. Citation Format: Tareq Saleh, Moureq R. Alotaibi, Sarah L. Kyte, Emmanuel K. Cudjoe, Ajinkya Kawale, Jingwen Xu, Zeinab Elsayed, Joseph W. Landry, Scott C. Henderson, Vasily Yakovlev, Lynne W. Elmore, David A. Gewirtz. Therapy-induced senescence is reversible in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 459.

The potential antitumor activity of cannabinoid receptor agonists, such as the aminoalklylindole WIN55,212-2 (WIN2), has been studied extensively, but their potential interaction with conventional cancer therapies, such as radiation, remains unknown. In the present work, the influence of WIN2 on the antiproliferative activity of radiation in human (MCF-7 and MDA-MB231) and murine (4T1) breast cancer cells was investigated. The antiproliferative effects produced by combination of WIN2 and radiation were more effective than either agent alone. The stereoisomer of WIN2, WIN55,212-3 (WIN3), failed to inhibit growth or potentiate the growth-inhibitory effects of radiation, indicative of stereospecificity. Two other aminoalkylindoles, pravadoline and JWH-015 [(2-methyl-1-propyl-1H-indol-3-yl)-1-naphthalenyl-methanone], also enhanced the antiproliferative effects of radiation, but other synthetic cannabinoids (i.e., nabilone, CP55,940 [(+)-rel-5-(1,1-dimethylheptyl)-2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]-phenol], and methanandamide) or phytocannabinoids [i.e., Δ9-tetrahydrocannabinol (THC) and cannabidiol] did not. The combination treatment of WIN2 + radiation promoted both autophagy and senescence but not apoptosis or necrosis. WIN2 also failed to alter radiation-induced DNA damage or the apparent rate of DNA repair. Although the antiproliferative actions of WIN2 were mediated through noncannabinoid receptor-mediated pathways, the observation that WIN2 interfered with growth stimulation by sphingosine-1-phosphate (S1P) implicates the potential involvement of S1P/ceramide signaling pathways. In addition to demonstrating that aminoalkylindole compounds could potentially augment the effectiveness of radiation treatment in breast cancer, the present study suggests that THC and nabilone are unlikely to interfere with the effectiveness of radiation therapy, which is of particular relevance to patients using cannabinoid-based drugs to ameliorate the toxicity of cancer therapies.

Lung cancer is the leading cause of cancer-related death in both men and women in the United States. Most lung cancer cases are diagnosed in advanced, inoperable stages and are treated with chemoradiation; while chemoradiation is effective in suppressing tumor progression, recurrence following treatment is not infrequent. As there is no well-accepted preclinical model for tumor dormancy and disease recurrence, we hypothesize that the promotion of transient autophagy and senescence could be developed as a model of tumor growth and recovery following treatment. Studies were performed utilizing H460 NSCLC cells as well as a primary NSCLC cell line isolated and grown in our laboratory from a stage IV adenocarcinoma. After treatment with etoposide (1 uM), growth arrest was accompanied by the robust induction of autophagy and senescence. This growth arrest was transient and was followed by proliferative recovery in the course of several days. Genetic and pharmacological inhibition of autophagy failed to interfere with the ability of the cells to regrow, indicating a non-cytoprotective function of autophagy in response to etoposide (in contrast to the cytoprotective function of autophagy exhibited in response to radiation). Quantification of senescence over time based on C12FDG staining and flow cytometry demonstrated that the reversal of growth arrest coincided with a decline in the extent of senescence. To more precisely define the source of the recovered cells, senescent and non-senescent but growth arrested cells were separated by flow cytometry based on their relative β-galactosidase expression and replated. Both cell populations demonstrated the ability to re-emerge from the growth-arrested state and recover proliferative capacity. These observations suggest that senescence is ultimately a transient process in that at least a subpopulation of tumor cells can and will recover proliferative capacity. Furthermore, the reversibility of therapy-induced senescence (TIS) might prove to be a useful model both in cell culture and in vivo to study tumor dormancy and disease recurrence. Studies are currently in progress to determine the impact of senescence inhibition on tumor recovery as well as sensitivity to subsequent therapy given that recurrent disease tends to be relatively refractory to further treatment. Citation Format: Tareq Saleh, Theresa Thekkudan, Moureq R. Alotaibi, David A. Gewirtz. Proliferative recovery after chemotherapy-induced senescence in non-small cell lung cancer (NSCLC) cells as a model of tumor dormancy and disease recurrence. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2846.

Radiotherapy continues to be a primary modality in the treatment of cancer. DNA damage induced by radiation can promote apoptosis as well as both autophagy and senescence, where autophagy and senescence can theoretically function to prolong tumor survival. A primary aim of this work was to investigate the hypothesis that autophagy and/or senescence could be permissive for DNA repair, thereby facilitating tumor cell recovery from radiation-induced growth arrest and/or cell death. In addition, studies were designed to elucidate the involvement of autophagy and senescence in radiation sensitization by PARP inhibitors and the re-emergence of a proliferating tumor cell population. In the context of this work, the relationship between radiation-induced autophagy and senescence was also determined. Studies were performed using DNA repair proficient HCT116 colon carcinoma cells and a repair deficient Ligase IV (-/-) isogenic cell line. Irradiation promoted a parallel induction of autophagy and senescence that was strongly correlated with the extent of persistent H2AX phosphorylation in both cell lines; however inhibition of autophagy failed to suppress senescence, indicating that the two responses were dissociable. Irradiation resulted in a transient arrest in the HCT116 cells while arrest was prolonged in the Ligase IV (-/-) cells; however, both cell lines ultimately recovered proliferative function, which may reflect maintenance of DNA repair capacity. The PARP inhibitors (Olaparib) and (Niraparib) increased the extent of persistent DNA damage induced by radiation as well as the extent of both autophagy and senescence; neither cell line underwent significant apoptosis by radiation alone or in the presence of the PARP inhibitors. Inhibition of autophagy failed to attenuate radiation sensitization, indicating that autophagy was not involved in the action of the PARP inhibitors. As with radiation alone, despite sensitization by PARP inhibition, proliferative recovery was evident within a period of 10-20 days. While inhibition of DNA repair via PARP inhibition may initially sensitize tumor cells to radiation via the promotion of senescence, this strategy does not appear to interfere with proliferative recovery, which could ultimately contribute to disease recurrence. Citation Format: Moureq R. Alotaibi, Khushboo Sharma, Tareq Saleh, Lawrence Povirk, Eric Hendrickson, David Gewirtz. Autophagy, senescence and proliferative recovery subsequent to DNA damage in radiation sensitization by PARP inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2848.

Radiotherapy is used along with other modalities such as surgery, chemotherapy, and immunotherapy to either shrink tumors before surgery or kill surviving tumor cells post surgery. Although radiation (and chemotherapy) induce different modes of cell death, recurrence of cancer may occur. It has been shown that DNA damage can induce cells to undergo both autophagy and senescence where both responses can function either as a pro-survival mechanism or in promoting loss of self-renewal capacity in the tumor cell. The relationship between autophagy, senescence, and the DNA repair system has not been well-characterized. The primary aims of this work were to understand whether autophagy and senescence might be permissive for DNA repair, thereby allowing the tumor cells to overcome the persistent DNA damage and recover using HCT116 WT and HCT116 Ligase IV (-/-) human colon carcinoma cells. Irradiation caused HCT116 Ligase IV (-/-) cells to lose their capacity to proliferate followed by delayed cell death, whereas HCT116 WT cells showed proliferative recovery 72 hours post-treatment, consistent with deficiencies in DNA repair in the Ligase IV (-/-) cells. Irradiation of both cell lines resulted in enlargement in cell size and accumulation of acidic vacuoles, indicative of the induction of autophagy. Inhibition of autophagy by Bafilomycin and Choroquine resulted in radio-sensitization in HCT116 WT cells but not in HCT116 Ligase IV (-/-) cells, indicating that radiation-induced autophagy in HCT116 WT cells is likely to be cytoprotective. Both repair competent HCT116 colon carcinoma cells and Ligase IV deficient cells demonstrated significant increases in senescent populations upon irradiation based on β-galactosidase staining and enlargement of treated cells. The PARP inhibitor, Olaparib, enhanced the efficacy of radiation through interference with DNA repair and the promotion of both autophagy and senescence but not apoptosis. Taken together, it appears that both autophagy and senescence may facilitate DNA repair during exposure to genotoxic stress, and that autophagy and senescence are closely linked responses in irradiated tumor cells. Citation Format: Moureq R. Alotaibi, Lawrence Povirk, Daivd Gewirtz. The relationship between autophagy, senescence, and DNA repair. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 993. doi:10.1158/1538-7445.AM2015-993

Radiotherapy is used along with other modalities such as surgery, chemotherapy, and immunotherapy to either shrink tumors before surgery or kill surviving tumor cells post surgery. Although radiation may induce different modes of cell death, recurrence of cancer may occur. It has been shown that DNA damage can induce cells to undergo both autophagy and senescence where both responses can function either as a pro-survival mechanism or in promoting loss of self-renewal capacity in the tumor cell. The relationship between autophagy, senescence, and the DNA repair system has not been well-characterized. The primary aims of this work were to understand whether autophagy and senescence might be permissive for DNA repair thereby allowing the tumor cells to overcome the persistent DNA damage and recover. Studies were performed using M059K (DNA-PK proficient) and M059J (DNA-PK deficient) human glioma cells to assess the kinetics of DNA repair after radiation in the absence and presence of autophagy inhibitors. M059J cells were observed to lose their capacity to proliferate as they undergo growth arrest followed by delayed cell death, whereas M059K cells showed proliferative recovery 72 hours post-treatment. Both irradiated cells showed an enlargement in cell size and accumulation of acidic vacuoles, indicating the induction of autophagy. Both cell lines also demonstrated reduced p62 level after radiation, indicative of autophagic flux. Inhibition of autophagy by 3-MA showed a radio-sensitization in M059K cells but not in M059J cells, indicating that radio-induced autophagy in M059K cells might be cytoprotective. Additional studies were performed in HCT116 cells that lack ligase IV to demonstrate a correspondence between the extent of drug or radiation induced senescence and the persistence of DNA damage foci. Ligase IV deficient cells were more sensitive to low radiation doses (2 Gy), whereas the ligase proficient cells showed recovery 96 hrs post-treatment. Both parental HCT116 colon carcinoma cells and Ligase IV deficient cells demonstrated significant increases in senescent populations at low doses of radiation based on β-galactosidase staining and enlargement and extensions of treated cells; senescence was more pronounced in the Ligase IV deficient cells. HCT116 cells were then treated with a dose of radiation (8 Gy) that induces ∼75% of senescence after 96 hrs and then reirradiated with half of that dose (4 Gy) to monitor the ability of senescent cells to repair the DNA damage. Although the majority of the cells were in a state of senescence, we found that these cells were able to repair the DNA damage induced by the second dose.Taken together, it seems that autophagy has an important function in the enhancement of DNA repair during exposure to genotoxic stress, but the role of autophagy may differ according to the status of DNA repair. Also, persistent DNA damage could be a determinant for senescence, and senescent cells might be able to repair these breaks. Citation Format: Moureq R. Alotaibi, Daivd Gewirtz, Lawrence Povirk. Involvement of autophagy and senescence in DNA repair in irradiated tumor cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2385. doi:10.1158/1538-7445.AM2014-2385

The stilbene derivative, cis-3, 4’, 5-trimethoxy-3’-aminostilbene (stilbene 5c), is a potentially potent antitumor agent that acts via binding to the colchicine-binding pocket in microtubules. Earlier studies have shown that stilbene 5c induces cell death in ovarian cancer cells and leukemic cells (Cao TM et al. Am J Hematol. 2008;83:390-7; Durrant D et al. Gynecol Oncol. 2008; 110:110-7).). The present study was designed to investigate the effectiveness of this microtubule poison against the HCT-116 human colon cancer cell line as well as B16F10 melanoma cells and its mechanisms of action. Time course studies demonstrated that stilbene 5c produces a decrease in cell viability in both cell lines. The capacity of the cells to proliferate was not restored upon removal of the drug after 5 days of exposure. Consistent with the results of the time course studies, β-galactosidase staining indicated that treatment with stilbene 5c also promotes senescence. In addition to senescence, HCT-116 cells and B16F10 melanoma cells treated with stilbene 5c displayed formation of autophagic vesicles by acridine orange staining, which was supported by fluorescence-activated cell sorting (FACS). Further evidence of autophagy was derived from treatment of HCT116 cells expressing an RFP-LC3 construct with stilbene 5c, in which LC3 puncta formation increased in a time-dependent manner. DAPI staining, TUNEL, and Annexin 5 staining indicated that apoptosis is also occurring in stilbene 5c-treated HCT-116 cells. Cell cycle analysis of HCT116 cells demonstrated growth arrest at both G1 and G2/M, and an increase in the subG1 population at days 3 and 5, which correspond to senescence and apoptosis respectively. Interestingly, DAPI and Hoechst staining of HCT116 cells revealed morphological changes in the cell nuclei (binucleated and micronucleated cells), which suggest that mitotic catastrophe may also serve as a mode of cell death after treatment with stilbene 5c. Our studies also indicated that stilbene 5c works in a p53-independent manner. p53-null HCT116 cells demonstrated similar sensitivity to stilbene as p53-wild type HCT116 cells. Consistent with previous studies in other experimental cancer models, this work indicates that stilbene 5c could potentially be effective against melanoma and colon cancer through the promotion of multiple modes of cell death. Citation Format: Moureq R. Alotaibi. Cell death and growth arrest pathways mediating the actions of stilbene 5c in HCT-116 colon cancer cells and B16F10 melanoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1658. doi:10.1158/1538-7445.AM2013-1658

The stilbene derivative, cis-3, 4′, 5-trimethoxy-3′-aminostilbene (stilbene 5c), is a potentially potent antitumor agent that acts via binding to the colchicine-binding pocket in microtubules. Earlier studies have shown that stilbene 5c induces cell death in ovarian cancer cells and leukemic cells (Lee RM et al, 2008; Lee RM et al, 2008). The present study was designed to investigate the effectiveness of this microtubule poison against the HCT-116 human colon cancer cell line and its mechanisms of action. Time course studies demonstrated that stilbene 5c produces a biphasic decrease in cell viability. The capacity of the cells to proliferate was not restored upon removal of the drug after 6 days of exposure. Consistent with the results of the time course studies, β-galactosidase staining indicated that treatment with stilbene 5c also promotes senescence. In addition to senescence, stilbene 5c-treated HCT-116 cells displayed formation of autophagic vesicles by acridine orange staining, which was supported by fluorescence-activated cell sorting (FACS). Further evidence of autophagy was derived from treatment of HCT116 cells carrying an RFP-LC3 construct with stilbene 5c, in which LC3 puncta formation increased in a time-dependent manner. DAPI staining, TUNEL, and Annexin 5 staining indicated that apoptosis is also occurring in stilbene 5c-treated HCT-116 cells. Cell cycle analysis demonstrated growth arrest at both G1 and G2/M, and an increase in the subG1 population at days 3 and 5, which correspond to senescence and apoptosis respectively. Interestingly, DAPI and Hoechst staining revealed morphological changes in the cell nuclei (binucleated and micronucleated cells), which suggest that mitotic catastrophe may also serve as a mode of cell death after treatment with stilbene 5c. Consistent with previous studies in other experimental cancer models, this work indicates that stilbene 5c could potentially be effective against colon cancer through the promotion of multiple modes of cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3957. doi:1538-7445.AM2012-3957