Apoptosis-inducing Agents Research Articles

Synthesis and in vitro anticancer activity of certain novel 1-(2-methyl-6-arylpyridin-3-yl)-3-phenylureas as apoptosis-inducing agents

In connection with our research program on the development of novel anticancer candidates, herein we report the design and synthesis of novel series of 1-(2-methyl-6-arylpyridin-3-yl)-3-phenylureas 5a–l. The target pyridins were evaluated for their in vitro anticancer activity against two cancer cell lines: non-small cell lung cancer A549 cell line and colon cancer HCT-116 cell line. Compound 5l emerged as the most active congener towards both A549 and HCT-116 cell lines with IC50 values equal to 3.22 ± 0.2 and 2.71 ± 0.16 µM, respectively, which are comparable to those of Doxorubicin; 2.93 ± 0.28 and 3.10 ± 0.22, respectively. Furthermore, compound 5l stood out as the most potent pyridine derivative (mean % GI = 40), at US-NCI Developmental Therapeutic Program anticancer assay, with broad-spectrum antitumor activity against the most tested cancer cell lines from all subpanels. Compound 5l was able to provoke apoptosis in HCT-116 cells as evidenced by the decreased expression of the anti-apoptotic Bcl-2 protein, and the enhanced expression of the pro-apoptotic proteins levels; Bax, cytochrome C, p53, caspase-3 and caspase-9. Moreover, 5l disrupted the HCT-116 cell cycle via alteration of the Sub-G1 phase and arresting the G2-M stage. Also, 5l showed a significant increase in the percent of annexinV-FITC positive apoptotic cells from 1.99 to 15.76%.

Bacteriophage MS2 As a Tool for Targeted Delivery in Solid Tumor Chemotherapy.

Bacteriophage MS2 was employed for targeted delivery of an apoptosis-inducing agent, Tl+, into a tumor tissue. The targeted delivery was ensured by iRGD peptide, a ligand of integrins presumably located on the surface of endotheliocytes of the tumor tissue neovasculature and certain tumor cells. The synthesized peptide was conjugated to MS2 capsid proteins. Tl+ ions from TlNO3 penetrated the phage particles and tightly bound to phage RNA. Peptide-modified MS2 preparations filled with Tl+ caused cell death in two types of cultivated human breast cancer cells and effected necrosis of these tumor xenografts in mice. Neither peptide-conjugated bacteriophage MS2 without Tl+ nor the phage filled with Tl+ but without the peptide or the same phage with the non-conjugated peptide in solution produced such effects. The preparation exhibited no acute toxicity at a therapeutic dose.

A new insight into the apoptotic effect of nitidine chloride targeting Checkpoint kinase 2 in human cervical cancer in vitro.

Nitidine chloride (NC), a natural, bioactive, phytochemical alkaloid derived from the roots of Zanthoxylum nitidum, has been reported to exhibit anti-tumor activity against various types of cancer. However, the potential therapeutic role of NC in human cervical cancer has not yet been studied. We are the first to report that NC acts as a potential apoptosis-inducing agent for human cervical cancer in vitro. NC treatment of human cervical cancer cell lines induced caspase-mediated apoptosis, thereby reducing cell viability. Phospho-kinase proteome profiling using a human phospho-kinase array revealed that NC treatment phosphorylated Checkpoint kinase 2 (Chk2) at Thr68, which activates Chk2 in both cell lines. We also found that NC significantly affected the p53/Bim signaling axis, which was accompanied by mitochondrial membrane depolarization and cytochrome c release from the mitochondria into the cytosol. In addition, NC profoundly increased phosphorylation of the histone variant H2AX at Ser139, a typical marker of DNA damage. Taken together, these results provide in vitro evidence that NC can increase Chk2 activation, thereby acting as an attractive cell death inducer for treatment of human cervical cancer.

Curcuminoid B63 induces ROS-mediated paraptosis-like cell death by targeting TrxR1 in gastric cells

Gastric cancer is one of the leading causes of cancer-related deaths. Chemotherapy has improved long-term survival of patients with gastric cancer. Unfortunately, cancer readily develops resistance to apoptosis-inducing agents. New mechanisms, inducing caspase-independent paraptosis-like cell death in cancer cells is presently emerging as a potential direction. We previously developed a curcumin analog B63 as an anti-cancer agent in pre-clinical evaluation. In the present study, we evaluated the effect and mechanism of B63 on gastric cancer cells. Our studies show that B63 targets TrxR1 protein and increases cellular reactive oxygen species (ROS) level, which results in halting gastric cancer cells and inducing caspase-independent paraptotic modes of death. The paraptosis induced by B63 was mediated by ROS-mediated ER stress and MAPK activation. Either overexpression of TrxR1 or suppression of ROS normalized B63-induced paraptosis in gastric cancer cells. Furthermore, B63 caused paraptosis in 5-fluorouracil-resistant gastric cancer cells, and B63 treatment reduced the growth of gastric cancer xenografts, which was associated with increased ROS and paraptosis. Collectively, our findings provide a novel strategy for the treatment of gastric cancer by utilizing TrxR1-mediated oxidative stress generation and subsequent cell paraptosis.

Design and synthesis of some β-carboline derivatives as multi-target anticancer agents.

Some anticancer β-carbolines exhibited dual inhibition of topo-I and KSP. Novel β-carbolines were synthesized and screened for their anticancer activity according to the NCI protocol. Five dose assays results indicated that compounds 9, 10, 12, 17 and 20 were potent and non selective anticancer agents; the sulfanyltriazole 12 was the most potent. Compounds 10, 12 and 20 showed dual topo-I and KSP inhibition with compound 12 being the most potent. Active compounds elicited Pre-G1 apoptosis and cell cycle arrest at G2/M phase of melanoma MDA-MB-435 cells. Docking results, in silico physicochemical and absorption, distribution, metabolism, excretion (ADME) properties were appropriate. Compounds 10, 12 and 20 are potent apoptosis-inducing multitarget anticancer agents that act via dual inhibition of topo-I and KSP-ATPase.

A Multi-Drug Tolerant Phenotype Is Induced By Diverse Microenvironmental Agonists in Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma

Responses to single-agent ibrutinib (IBR) or venetoclax (VEN) in Chronic Lymphocytic Leukemia (CLL) or Mantle Cell Lymphoma (MCL) are often incomplete suggesting drug combinations are needed to overcome de novo and acquired resistance. We previously reported synergistic cytotoxicity for the IBR+VEN combination in CLL/MCL cells ex vivo and have initiated an IBR+VEN clinical trial in MCL (NCT02419560). However, ex vivo analysis of patient samples showed de novo resistance even to the IBR+VEN combination in some samples, consistent with recent clinical experience (Tam et al. N Engl J Med, 2018). We noted that sensitivity to IBR+VEN was lower in CLL cells showing an “activation” phenotype (CD5+/19+/69+) that can be induced by extracellular factors in vivo, suggesting that the microenvironment could induce drug tolerance in the cancer cells.This was supported by our finding that ex vivo exposure of CLL/MCL samples to a mixture of microenvironmental agonists (CpG-ODN, sCD40L, and IL10; “agonist mix”) induced significant loss of sensitivity to IBR+VEN (Jayappa et al. Blood Adv., 2017). Here we show that various microenvironmental agonists, including of innate immunity, are able to reduce sensitivity to IBR+VEN as well as other pro-apoptotic drugs, generating a multi-drug tolerant phenotype.To explore drug tolerance, we performed flow cytometric analysis of apoptosis induced by IBR+VEN in CD5+/19+cancer cells in CLL/MCL PBMCs cultured with agonists of TLRs, NOD1/2, CD40, and IL10R and various stromal cells. Tolerance to IBR+VEN as determined by resistance to apoptosis was noted in most samples cultured with TLR9 agonist CpG-ODN, sCD40L, or Jurkat cells expressing CD40L. IL10 and HUVEC induced modest levels of drug tolerance in a few CLL/MCL samples, and TLR1/2, TLR7, and NOD1/2 agonists were effective only in MCL samples.Prior exposure to CpG-ODN enhanced the ability of sCD40L to induce proliferation and drug tolerance and vice versa in CLL/MCL, predicting mutually reinforcing interactions in vivo. We noted that CLL cells exposed to CpG-ODN displayed higher levels of CD40 and downstream signaling proteins (TRAF2, RelB, and p100/52). Conversely, CD40L induced a modest increase in NF-kB transcription factors, providing a possible mechanism for mutual reinforcement.Using flow cytometric analysis of apoptosis, we noted tolerance to several pro-apoptotic agents (bendamustine, fludarabine, and inhibitors of Mcl-1, Bcl-xL, Bcl-2, and Bcl-2/Bcl-xL) including IBR+VEN in CD5+/19+cells from CLL samples treated with agonist mix, showing development of multi-drug tolerance in these cells. CpG-ODN or agonist mix induced NFkB-dependent over-expression of pro-survival proteins (Mcl-1 and Bcl-xL), and increased ratio of these to cognate pro-apoptotic proteins. This increased expression of pro-survival proteins underlies the multi-drug tolerant phenotype. Consistently, multi-drug tolerance was rescued with inhibitors of NFkB (BMS345541 or Bortezomib) or drug combinations simultaneously inhibiting multiple anti-apoptotic proteins (inhibition of Mcl-1 with Bcl-2, Bcl-xL, or Bcl-2 and Bcl-xL).CLL cells with activation phenotype (CD5+/19+/69+) in patient PBMCs cultured without the agonist mix also showed tolerance to several apoptotic protein inhibitors ex vivo, and this was also effectively rescued with NF-kB inhibition or combination of apoptotic protein inhibitors ex vivo. These results suggest that the cancer cells with activation phenotype exist in vivo anddisplay multi-drug tolerant phenotype consistent with the drug tolerance induced by agonist mix ex vivo.In conclusion, several microenvironmental factors, particularly agonists of TLR9 and CD40, induce tolerance to IBR+VEN and cell proliferation in CLL/MCL, and response to these agonists is enhanced by combinatorial exposure. These agonists generate tolerance to several apoptosis-inducing agents beyond IBR+VEN. This microenvironment-induced multi-drug tolerance is mediated by NFkB dependent over-expression of multiple pro-survival proteins. Inhibitors of NFkB, or drug combinations targeting multiple pro-survival proteins, overcame multi-drug tolerance in agonist mix-treated samples and in CD69+CLL cells in patient PBMCs that represent multi-drug tolerant cells in vivo. Thus, more complete and durable responses might be achieved in MCL/CLL with therapies that target multi-drug tolerant persister cells. DisclosuresWilliams:Celgene: Consultancy, Research Funding; Kite: Consultancy; Juno: Consultancy; Seattle Genetics: Consultancy; Gilead: Consultancy, Research Funding; Novartis: Research Funding; TG Therapeutics: Consultancy; Sandoz: Consultancy; Astra-Zeneca: Consultancy; Abbvie: Consultancy; Takeda: Research Funding; Pharmacyclics: Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Verastem: Consultancy. Portell:AbbVie: Research Funding; Infinity: Research Funding; Genentech/Roche: Consultancy, Research Funding; Acerta: Research Funding; BeiGene: Research Funding; Kite: Research Funding; Amgen: Consultancy; TG therapeutics: Research Funding.

Synthesis and biological evaluation of 2-(3-aminophenyl)-benzothiazoles as antiproliferative and apoptosis-inducing agents

A series of new 2-(3-aminophenyl)-benzothiazole derivatives were synthesized and evaluated for their in vitro antiproliferative activity against various human cancer cell lines including A549, HeLa, HepG2, MCF-7, MV4-11, and DB. Among the tested compounds, N-[3-(benzo[d]thiazol-2-yl)phenyl]nicotinamide displayed significantly improved antiproliferative activity toward A549 and MV4-11 cells with IC50 values of 5.42 ± 1.33 and 7.51 ± 0.98 μM, respectively, much stronger than the hit 3-(benzo[d]thiazol-2-yl)-N-(4-bromobenzyl)aniline. Furthermore, flow cytometric analysis indicated that N-[3-(benzo[d]thiazol-2-yl)phenyl]nicotinamide induced A549 cell apoptosis with cell cycle arrest at G1 phase in a concentration-dependent manner.

Mitochondrial targeting domain of NOXA causes necrosis in apoptosis-resistant tumor cells.

The resistance of tumor cells to apoptosis-inducing anticancer agents is regarded as a major impediment for the treatment of cancer patients. This study aimed to examine the possibility whether a necrosis-inducing peptide containing the mitochondria-targeting domain (MTD) of NOXA kills tumor cells that are resistant to apoptosis-inducing anticancer agents. To examine this possibility, we established doxorubicin-resistant (Dox-Res) cells by treating CT26 cells with increasing amounts of doxorubicin. The apoptosis resistance of the Dox-Res CT26 cells was confirmed by measuring the cell viability and activation of caspases. We showed that the MTD-containing peptide fused to eight arginine residues (R8:MTD), a necrosis-inducing peptide, induced necrosis in the Dox-Res CT26 cells, together with a cytosolic calcium spike, reactive oxygen species production, and the release of high mobility group box 1 into the media. Moreover, we demonstrated the killing effect of R8:MTD in tumor tissues generated using the Dox-Res CT26 cells in a mouse model. Therefore, our results suggest that MTD-containing peptides may provide an alternative tool for the elimination of relapsed tumor cells that are not responsive to apoptosis-inducing anticancer agents.

Design, synthesis, cytotoxicity and molecular modeling studies of some novel fluorinated pyrazole-based heterocycles as anticancer and apoptosis-inducing agents.

3,5-Diamino-4-(3-trifluoromethylphenyldiazenyl)-1H-pyrazole was used as a starting scaffold for the synthesis of new pyrazole-based heterocycles to study their effects on the proliferation of three human cancer cell lines; human liver carcinoma cell line (HepG-2), colon cancer cell line (HCT-116) and human breast cancer cell line (MCF-7) using MTT assay. The synthesized compounds were characterized on the basis of IR, 1H NMR, 13C NMR, mass spectral data and elemental analysis results. Cytotoxicity assay results revealed that some of the compounds showed potent growth inhibition against all the cell lines tested, with IC50 values in the range of 0.64-7.73μg/mL. Breast cancer cells were used for further detailed studies to understand the mechanism of cell growth inhibition and apoptosis-inducing effect of the most active compounds. The results indicated that compounds 3a, 10b and 11a arrested MCF-7 cells at G2/M phase of the cell cycle and might induce apoptosis via caspase-3-dependent pathway. Molecular modeling and binding mode analysis of the most active compounds to caspase 3 active site further provide a synergistic mechanism for their pro-apoptotic effects. In order to explore the structural requirements controlling the observed cytotoxic properties, 3D pharmacophore model was generated.

Tissue-Nonspecific Alkaline Phosphatase Is Required for MC3T3 Osteoblast-Mediated Protection of Acute Myeloid Leukemia Cells from Apoptosis.

The bone marrow microenvironment harbors and protects leukemic cells from apoptosis-inducing agents via mechanisms that are incompletely understood. We previously showed SDF-1 (CXCL-12), a chemokine readily abundant within the bone marrow microenvironment, induces apoptosis in acute myeloid leukemia (AML) cells that express high levels of the SDF-1 receptor CXCR4. However, differentiating osteoblasts found within this niche protect cocultured AML cells from apoptosis. Additionally, this protection was abrogated upon treatment of the differentiating osteoblasts with histone deacetylase inhibitors (HDACi). In this study, we begin to characterize and target the molecular mechanisms that mediate this osteoblast protection. Quantitative RT-PCR revealed that HDACi treatment of differentiating osteoblasts (mouse MC3T3 osteoblast cell line) reduced expression of multiple genes required for osteoblast differentiation, including genes important for producing mineralized bone matrix. Interestingly, pretreating differentiating osteoblasts with cyclosporine A, a drug known to inhibit osteoblast differentiation, similarly impaired osteoblast-mediated protection of cocultured AML cells (KG1a and U937 human AML cell lines). Both HDACi and cyclosporine A reduced osteoblast expression of the key mineralization enzyme tissue-nonspecific alkaline phosphatase (TNAP; encoded by Alpl). Moreover, specifically reducing TNAP expression or activity in differentiating osteoblasts significantly impaired the ability of the osteoblasts to protect cocultured AML cells. Together, our results indicate that inhibiting osteoblast matrix mineralization by specifically targeting TNAP is sufficient to significantly impair osteoblast-mediated protection of AML cells. Therefore, designing combination therapies that additionally target the osteoblast-produced mineralized bone matrix may improve treatment of AML by reducing the protection of leukemic cells within the bone marrow microenvironment.

Abstract 4823: Alvocidib enhances the efficacy of cytarabine and daunorubicin (7+3) in non-clinical models of acute myeloid leukemia

Abstract The ‘7+3' induction regimen has been the standard of care for frontline acute myeloid leukemia (AML) for more than 30 years. There are still significant unmet needs for frontline patients, particularly those with high-risk features. Alvocidib, a potent CDK9 inhibitor, has shown clinical activity in multiple studies of frontline and relapsed/refractory AML. In some clinical studies, alvocidib has been administered as part of the ACM (alvocidib, cytarabine, and mitoxantrone) regimen. The ACM regimen has demonstrated greater clinical responses compared to 7+3 in frontline intermediate and high-risk AML patients. The ACM regimen is now being evaluated in a multi-institution, Phase II study using a MCL-1 dependency biomarker to select patients likely to be sensitive to the alvocidib-containing regimen. Alvocidib activity is primarily driven by its inhibition of CDK9/RNA-polymerase II and the subsequent suppression of key anti-apoptotic proteins, including MCL-1. Alvocidib induces apoptosis and potentiates the activity of apoptosis-inducing agents, such as cytarabine and mitoxantrone. The synergy of the ACM regimen led us to hypothesize that alvocidib may enhance the anti-leukemia activity of the 7+3 regimen in preclinical models as well. Cell viability and apoptosis measurements were made following treatment using the CellTiter-Glo and Caspase-Glo assays according to manufacturer protocol. MCL-1 mRNA expression and the expression of additional markers was determined using RT-qPCR. Protein expression was determined using standard immunoblotting techniques. Alvocidib and the 7+3 regimen were also tested in vivo in the MV4-11 AML xenograft model. Here, it was observed that the 72-hour viability assays yielded single agent IC50 values of alvocidib, cytarabine, or daunorubicin in cultured AML cells ranging from 2.2 nM to 567 nM. In apoptosis assays, a modest induction was observed with cytarabine, daunorubicin, or alvocidib. However, the three-drug combination more than doubled the induction of apoptosis compared to any of the agents alone. We also observed that MCL-1 was suppressed in the combination treatment. In a xenograft study, 21.1 and 48.5% tumor growth inhibition (TGI) was observed following either single agent treatment of daunorubicin (0.5 mg/kg) or cytarabine (60 mg/kg), respectively. Treatment of alvocidib (1.25 mg/kg) yielded 60.0% TGI. Combination treatment, however, resulted in tumor shrinkage, yielding 116.2% TGI. While we have reported previously that alvocidib synergizes with cytarabine and mitoxantrone preclinically in the ACM regimen, our data here demonstrates that alvocidib potentiates the activity of cytarabine and daunorubicin as well. Our results here provide additional scientific rationale for an investigational Phase Ib clinical study employing alvocidib in combination with 7+3 in frontline AML patients, which is currently ongoing. Citation Format: Wontak Kim, Clifford J. Whatcott, Adam Siddiqui-Jain, David J. Bearss, Steven L. Warner. Alvocidib enhances the efficacy of cytarabine and daunorubicin (7+3) in non-clinical models of acute myeloid leukemia [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 4823.

Effects of glaucocalyxin A on human liver cancer cells as revealed by GC/MS- and LC/MS-based metabolic profiling.

Studies have documented the potential antitumor activities of glaucocalyxin A (GLA), an ent-kaurene diterpenoid isolated from Rabdosia japonica. However, the metabolic mechanism underlying the antitumor activity of GLA remains largely unknown. The effects of GLA on the metabolome of human liver cancer cells using GC/MS- and LC/MS-based metabolic profiling have been investigated. An untargeted metabolomics approach in conjunction with orthogonal projection to latent structures-discriminant analysis (OPLS-DA) has been developed to characterize the metabolic modifications induced by GLA treatment in human hepatoma cell line SMMC7721. Results demonstrated that cells cultured in the presence or absence of GLA displayed different metabolic profiles: the treatment induced an increased purine metabolism, pyrimidine metabolism, and sphingolipid metabolism and a decreased amino acid metabolism. At the same time, GLA treatment induced cell apoptosis and cell cycle arrested at G2/M phase in a dose-dependent manner. In addition, two representative apoptosis-inducing cytotoxic agents were selected as positive control drugs to validate the reasonableness and accuracy of our metabolomic investigation on GLA. The study displayed a systemic metabolic alteration induced by GLA treatment, showing the impaired physiological activity of SMMC7721 cells, which also indicated anti-proliferative and apoptotic effects of GLA. In the meantime, GC/MS- and LC/MS-based metabolomics applied to cell culture enhanced our current understanding of the metabolic response to GLA treatment and its mechanism; such an approach could be transferred to study the mechanism of other anticancer drugs. Graphical abstract A systemic metabolic alteration induced by glaucocalyxin A (GLA) treatment of SMMC-7721 cells.

Total Synthesis of Desmethyl Jahanyne and Its Lipo-Tetrapeptide Conjugates Derived from Parent Skeleton as BCL-2-Mediated Apoptosis-Inducing Agents.

The total synthesis of highly potent and scarcely available marine natural product (−)-jahanyne was attempted resulting in a solution-phase synthesis of pruned versions with comparable activity. A simple and facile synthetic route was employed for the preparation of pruned congeners and would be scalable. The lipophilic tail of the natural product was synthesized from R-(+)-citronellol, utilizing easily available chemicals. All the synthesized compounds were screened for apoptotic activity against a panel of cell lines. These compounds depicted marked binding to B cell lymphoma 2 till 50 °C in cellular thermal shift analysis.

Induction of Apoptosis in Metastatic Breast Cancer Cells: XV. Downregulation of DNA Polymerase-α - Helicase Complex (Replisomes) and Glyco-Genes.

In normal and cancer cells, successful cell division requires accurate duplication of chromosomal DNA. All cells require a multiprotein DNA duplication system (replisomes) for their existence. However, death of normal cells in our body occurs through the apoptotic process. During apoptotic process several crucial genes are downregulated with the upregulation of caspase pathways, leading to ultimate degradation of genomic DNA. In metastatic cancer cells (SKBR-3, MCF -7, and MDA-462), this process is inhibited to achieve immortality as well as overexpression of the enzymes for the synthesis of marker molecules. It is believed that the GSL of the lacto family such as LeX, SA-LeX, LeY, Lea, and Leb are markers on the human colon and breast cancer cells. Recently, we have characterized that a few apoptotic chemicals (cis-platin, L-PPMP, D-PDMP, GD3 ganglioside, GD1b ganglioside, betulinic acid, tamoxifen, and melphalan) in low doses kill metastatic breast cancer cells. The apoptosis-inducing agent (e.g., cis-platin) showed inhibition of DNA polymerase/helicase (part of the replisomes) and also modulated (positively) a few glycolipid-glycosyltransferase (GSL-GLTs) transcriptions in the early stages (within 2h after treatment) of apoptosis. These Lc-family GSLs are also present on the surfaces of human breast and colon carcinoma cells. It is advantageous to deliver these apoptotic chemicals through the metastatic cell surfaces containing high concentration of marker glycolipids (Lc-GSLs). Targeted application of apoptotic chemicals (in micro scale) to kill the cancer cells would be an ideal way to inhibit the metastatic growth of both breast and colon cancer cells. It was observed in three different breast cancer lines (SKBR-3, MDA-468, and MCF-7) that in 2h very little apoptotic process had started, but predominant biochemical changes (including inactivation of replisomes) started between 6 and 24h of the drug treatments. The contents of replisomes (replisomal complexes) during induction of apoptosis are not known. It is known that DNA helicase activities (major proteins catalyze the melting of dsDNA strands) change during apoptotic induction process. Previously DNA Helicase-III was characterized as a component of the replication complexes isolated from carcinoma cells and normal rapid growing embryonic chicken brain cells. Helicase activities were assayed by a novel method (combined immunoprecipitation-ROME assay), and DNA polymerase-alpha activities were determined by regular chain extension of nicked "ACT-DNA," by determining values obtained from +/- aphidicolin added to the incubation mixtures. Very little is known about the stability of the "replication complexes" (or replisomes) during the apoptotic process. DNA helicases are motor proteins that catalyze the melting of genomic DNA during replication, repair, and recombination processes. In all three breast carcinoma cell lines (SKBR-3, MCF-7, and MDA-468), a common trend, decrease of activities of DNA polymerase-alpha and Helicase-III (estimated and detected with a polyclonal antibody), was observed, after cis-platin- and L-PPMP-induced apoptosis. Previously our laboratory has documented downregulation (within 24-48h) of several GSL-GLTs with these apoptotic reagents in breast and colon cancer cells also. Perhaps induced apoptosis would improve the prognosis in metastatic breast and colon cancer patients.

Caspase-dependent apoptosis induced by two synthetic halogenated flavanones, 3',7-dichloroflavanone and 3',6-dichloroflavanone, on human breast and prostate cancer cells.

The destruction of cancer cells with chemotherapeutic agents is normally achieved through apoptosis. We previously introduced two synthetic halogenated flavanone derivatives, 3',7-dichloroflavanone (3'-7 DCF) and 3',6-dichloroflavanone (3'-6 DCF), as potential apoptosis-inducing agents. In the current study, we investigated the ability of these compounds in triggering intrinsic or/and extrinsic pathway of apoptosis in breast and prostate cancer cells. Also, the synergistic effect of 3'-7 DCF with TLR3 (Toll-like receptor 3) agonist in apoptosis induction was evaluated on PC3 and LNCaP human prostate cancer cells. The involved pathway of apoptosis in the treated cells was delineated by caspase-3 activity assay, PARP-1 (poly(ADP-ribose)polymerase-1) cleavage, and procaspase-9 cleavage as markers of the intrinsic pathway and procaspase-8 cleavage as the marker of the extrinsic pathway. With the exception of the normal cells, treatment of all cell lines with both 3'-7 DCF and 3'-6 DCF triggered the cleavage of procaspase-8 and procaspase-9. These results indicate that the intrinsic and the extrinsic pathways of apoptosis are the mechanisms of the toxicity of flavanones in these cancer cell lines. However, the cytoxicity of the compound 3'-7 DCF was not synergistic with TLR3 agonist. Interestingly, the activation of caspases-9 preceeded that of caspase-8 suggesting that the intrinsic pathway is the primary reason for apoptosis induction by the flavanones.

Apoptosis induction by alantolactone in breast cancer MDA-MB-231 cells through reactive oxygen species-mediated mitochondrion-dependent pathway.

Alantolactone is a sesquiterpene lactone isolated from Inula helenium L. Although alantolactone possesses anti-inflammation and apoptosis-induction activities, the underlying mechanism of anti-cancer effect on human breast cancer cells remains largely unknown. In this study, we explored the possibility of alantolactone as an apoptosis-inducing cytotoxic agent using MDA-MB-231 cells as in vitro model. Alantolactone significantly induced its apoptosis, demonstrated by cell cycle analysis, annexin V-APC/7-AAD double staining and dUTP nick end labeling. Additionally, alantolactone triggered the mitochondrial-mediated caspase cascade apoptotic pathway, which was confirmed by increased Bax/Bcl-2 ratio, loss of MMP, release of cytc from mitochondria to cytoplasm, activation of caspase 9/3, and subsequent cleavage of PARP. Z-VAD-FMK partially prevented apoptosis induced by alantolactone. Alantolactone provoked the production of ROS, while NAC (a scavenger of ROS) reversed alantolactone-mediated depolarization of MMP and apoptosis. Alantolactone modulated the activities of MAPKs. As expected, cotreatment with SB203580, SP600125 or U0126 could reduced the apoptotic rate. Furthermore, alantolactone decreased the protein expressions of p-NF-kB p65 and p-STAT3, increased p-c-Jun level in a dose-dependent manner. These findings suggested that alantolactone possessed anticancer activity via ROS-mediated mitochondrial dysfunction involving MAPK pathway, and had an effect on the transcription factors of NF-kB, AP-1 and STAT3.

Formulation, physical, in vitro and ex vivo evaluation of nanomedicine-based chemosterilant for non-surgical castration of male animals

The overpopulation of free-roaming companion animals has become the global crisis. The development and application of a suitable, effective, non-surgical approach for animal sterilization would have an enormous advantage over the current surgical method. The main purpose of this study was to develop and evaluate a novel nanomedicine-based chemosterilant for non-surgical castration of male companion animals. In this study, we first sought to investigate the testicular toxicity of different apoptosis-inducing agents. We next synthesized and characterized nano-sized particles which encapsulated the most potent testicular toxicants and evaluated in vitro sterilant properties. Our result showed that doxorubicin exhibited the highest cytotoxic activity against mouse spermatogenic cells. We therefore synthesized and characterized doxorubicin-encapsulated nanoemulsion. The negatively charged particle of doxorubicin-encapsulated nanoemulsion exhibited the anti-proliferative activity towards spermatogetic cells. Apoptosis studies revealed activation of Caspases 3 and 7 as well as annexin V expression. In addition, doxorubicin-encapsulated nanoemulsion exhibited anti-inflammatory activity in lipopolysaccharide-stimulated macrophages. Cell death was observed following treatment of isolated and cultured rat seminiferous tubules with doxorubicin-encapsulated nanoemulsion. In conclusion, nanoemulsion can be a potential carrier for prolonged release and to enhance activity of doxorubicin that may have utility in non-surgical castration of male animals.

Found in Translation: How Preclinical Research Is Guiding the Clinical Development of the BCL2-Selective Inhibitor Venetoclax.

Since the discovery of apoptosis as a form of programmed cell death, targeting the apoptosis pathway to induce cancer cell death has been a high-priority goal for cancer therapy. After decades of effort, drug-discovery scientists have succeeded in generating small-molecule inhibitors of antiapoptotic BCL2 family proteins. Innovative medicinal chemistry and structure-based drug design, coupled with a strong fundamental understanding of BCL2 biology, were essential to the development of BH3 mimetics such as the BCL2-selective inhibitor venetoclax. We review a number of preclinical studies that have deepened our understanding of BCL2 biology and facilitated the clinical development of venetoclax.Significance: Basic research into the pathways governing programmed cell death have paved the way for the discovery of apoptosis-inducing agents such as venetoclax, a BCL2-selective inhibitor that was recently approved by the FDA and the European Medicines Agency. Preclinical studies aimed at identifying BCL2-dependent tumor types have translated well into the clinic thus far and will likely continue to inform the clinical development of venetoclax and other BCL2 family inhibitors. Cancer Discov; 7(12); 1376-93. ©2017 AACR.

Inhibition of autophagy in hepatocarcinoma cells promotes chemotherapeutic agent-induced apoptosis during nutrient deprivation.

Autophagy is a lysosome-dependent process involved in protein and organelle degradation. It has been suggested that autophagy is activated in nutrient-deficient condition and plays an important role in protecting cells from nutrient shortage. However, the effect of autophagy on chemotherapy during nutrient deficiency has been rarely researched. In the present study, we discovered that hepatocarcinoma cells exhibit chemoinsensitivity accompanied by the activation of autophagy when cultured in nutrient-deprived medium. Inhibition of autophagy by 3-methyladenine or siRNA‑targeted Beclin1 increased the nutrient deprivation‑induced apoptosis and chemosensitivity in hepatocarcinoma cells. Furthermore, decreased mitochondrial mass was detected when cells underwent autophagy. The present study suggests that induction of autophagy confers a survival advantage for hepatocarcinoma cells during nutrient deprivation, not only rescuing cells from nutrient deficiency-induced cell apoptosis, but also protecting cells from chemotherapy-induced cell death. Combined usage of the inhibition of autophagy and conventional chemotherapeutic agents could be an effective therapy for hepatocarcinoma during nutrient deprivation.

Identification of 7,8-Diacetoxy-3-Arylcoumarin Derivative as a Selective Cytotoxic and Apoptosis-inducing Agent in a Human Prostate Cancer Cell Line.

Coumarins are a member of the benzopyrone family of compounds with diverse and interesting pharmacological properties. In the present study, we report the in vitro cytotoxicity evaluation of 7,8-Diacetoxy-3-arylcoumarin derivatives (5a-h) in human prostate (PC-3) and breast (MDA-MB-231) cancer cell lines. The cytotoxic activity was evaluated using crystal violet dye-binding assay. Furthermore, the most active compound in vitro cytotoxic activity in human non-cancerous cell line and its effect on the cell-cycle phases, apoptosis proteins expression, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) production and Glutathione (GSH) level were performed. Among the eight compounds that were evaluated, 7,8-Diacetoxy-3-(4-methylsulfonyl phenyl)coumarin (5f) was the most active derivative with highest cytotoxic activity and selectivity against the PC-3 cell line vs. the non-cancerous WPE1-N22 cell line. The cytotoxic action of compound 5f in PC-3 cells is associated with the cell-cycle arrest at -G0/G1 phase, apoptosis, loss in mitochondrial membrane potential (MMP), induced reactive oxygen species (ROS) production and depletion of Glutathione (GSH) level. The result indicates that the presence of p-methylsulfonylphenyl group on compound 5f is critical in modulating selective cytotoxic activity and induction of apoptosis via the mitochondrial apoptotic signaling pathway that is independent of cytochrome c release.