Combretastatin A-4 inhibits cell growth and metastasis in bladder cancer cells and retards tumour growth in a murine orthotopic bladder tumour model

BACKGROUND AND PURPOSE

Bladder cancer is a highly recurrent cancer after intravesical therapy, so new drugs are needed to treat this cancer. Hence, we investigated the anti-cancer activity of combretastatin A-4 (CA-4), an anti-tubulin agent, in human bladder cancer cells and in a murine orthotopic bladder tumour model.

EXPERIMENTAL APPROACH

Cytotoxicity of CA-4 was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, propidium iodide (PI) staining assay and clonogenic survival assay. In vivo microtubule assembly assay, cell cycle analyses, Western blot and cell migration assay were used to study the mechanism of CA-4. The effect of intravesical CA-4 therapy on the development of tumours was studied in the murine orthotopic bladder tumour model.

KEY RESULTS

CA-4 inhibited microtubule polymerization in vivo. Cytotoxic IC₅₀ values of CA-4 in human bladder cancer cells were below 4 nM. Analyses of cell-cycle distribution showed CA-4 obviously induced G₂-M phase arrest with sub-G₁ formation. The analyses of apoptosis showed that CA-4 induced caspase-3 activation and decreased BubR1 and Bub3 in cancer cells. In addition to apoptosis, CA-4 was also found to induce the formation of multinucleated cells. CA-4 had a significantly reduced cell migration in vitro. Importantly, the in vivo study revealed that intravesical CA-4 therapy retarded the development of murine bladder tumours.

CONCLUSIONS AND IMPLICATIONS

These data demonstrate that CA-4 kills bladder cancer cells by inducing apoptosis and mitotic catastrophe. It inhibited cell migration in vitro and tumour growth in vivo. Hence, CA-4 intravesical therapy could provide another strategy for treating superficial bladder cancers.     

Compound MMH01 possesses toxicity against human leukemia and pancreatic cancer cells

MMH01 is a compound isolated from Antrodia cinnamomea. MMH01 markedly inhibited growth of human leukemia U937 and pancreatic cancer BxPC3 cells. It resulted in distinct patterns of cell cycle distribution in U937 (G2/M, sub-G1 and polyploidy) and BxPC3 cells (G0/G1 and sub-G1). The modes of cell death in U937 cells include apoptosis and mitotic catastrophe, whereas apoptosis-associated events or necrosis in BxPC3 cells. Neither mitochondrial membrane permeabilization nor caspase dependence was noted. Proteins involving mitotic catastrophe-associated cell death such as cyclin B1 and checkpoint kinase 2 were activated in U937 cells. Only slight to moderate viability inhibition was noted to human monocytes, the normal counterpart of these myeloid leukemic cells. In conclusion, MMH01 possesses cytotoxicity against human leukemia and pancreatic cancer cells.     

Transcriptional changes facilitate mitotic catastrophe in tumour cells that contain functional p53

Exposure of Jurkat T lymphocytes containing functional p53 to nanomolar concentrations of bisanthracycline WP631 resulted in arrest at the G₂/M checkpoint and transient senescence-like phenotype in the presence of DNA synthesis. The cells entered crisis, became polyploid, showed aberrant mitotic figures, and died through mitotic catastrophe. Cell death was accompanied by changes in the expression profile of various oncogenes and tumour suppressor genes including the down-regulation of p53. The changed expression was confirmed for some of these genes using semi-quantitative RT-PCR, and the decline in p53 protein levels was established. Our results suggest that WP631 induced changes in cell cycle control pathways leading to death of Jurkat T cells through mitotic catastrophe, which occurred in the absence of caspase-2 and caspase-3 activities, rather than apoptosis.     

Genotoxicity of the marine toxin okadaic acid, in human Caco-2 cells and in mice gut cells

Micronucleus induction by the diarrhetic shellfish toxin okadaic acid (OA) was investigated in two intestinal models, cultured human Caco-2 cells and colon epithelial cells of mice treated in vivo. Exposure to OA for 4 and 24 h induced dose-responsive increases in the frequency of micronucleated Caco-2 cells; the minimum OA doses increasing micronucleus frequency were 20 nM for the 4 h treatment and 5 nM for the 24 h treatment. OA treatment of Caco-2 cells also resulted in dose- and time-dependent increases in mitotic arrest and multinucleated cells. Two experiments were conducted in which mice were treated with single oral gavages of 435-610 and 115-1341 microg/kg OA. In the first experiment, samples were taken 24 h after the treatment, and the frequencies of both micronucleated and mitotic gut cells were increased after treatment with 525 microg/kg OA. In the second experiment, no increases in micronucleus frequency were detected at 24, 36, or 48 h following OA doses of 230 and 115 microg/kg; however, an increase in the mitotic index was observed 36 h after a gavage with 115 microg/kg OA. In this experiment, doses higher than 230 microg/kg were rapidly lethal to the mice. Immunohistology with monoclonal OA antibodies showed that OA was distributed into the liver at all the sampling times and in the small intestine at 24 and 36 h; OA was not detected in the colon. In addition, the TUNEL assay indicated that OA induced apoptosis in mouse ileum, liver, and kidney. The results of our investigations suggest that OA is aneugenic in Caco-2 cells, whereas the in vivo data were inconclusive. Further studies should be performed in mice using intragastric doses of 230-525 microg/kg OA. Moreover, the apoptosis and cell proliferation results indicate that OA can reach organs other than colon, indicating further evaluation of the genotoxic potential of OA in these organs is warranted.     

Convergent synthesis and biological evaluation of 2-amino-4-(3',4',5'-trimethoxyphenyl)-5-aryl thiazoles as microtubule targeting agents

Combretastatin A-4, a potent tubulin polymerization inhibitor, caused us to synthesize a novel series of 2-amino-4-(3',4',5'-trimethoxyphenyl)-5-aryl thiazoles with the goal of evaluating the effects of substituents on the phenyl at the 5-position of the thiazole skeleton on biological activities. An ethoxy group at the para-position produced the most active compound in the series, with IC(50) values of 0.03-0.9 nM against five of seven cancer cell lines. The most active compounds retained full activity in multidrug resistant cancer cells and acted through the colchicine site of tubulin. Treated cells were arrested in the G2/M phase of the cell cycle, with cell death proceeding through an apoptotic pathway that was only partially caspase-dependent. Preliminary results suggest that, in addition to cell death by apoptosis, cells were also killed via mitotic catastrophe as an alternative cell death mechanism.     

Synthesis and preliminary biological evaluations of CC-1065 analogues: effects of different linkers and terminal amides on biological activity

CC-1065 analogues possessing a biologically active CBI functional group and amide-substituted indole and benzofuran were synthesized. The IC(50) values of compounds 26, bearing two indoles, and 25, bearing only one indole, are 0.4 and 3 nM, respectively, against U937 leukemia cells in vitro. The IC(50) values of compounds 28, bearing a butyramino group, and 27, bearing an acetamino group, are 0.008 and 0.4 nM, respectively, against U937 leukemia cells in vitro. Compound 29, bearing a double-bond linker, is about 4-fold more potent than 25, bearing no double-bond linker. Compound 26 is highly potent against all cell lines tested in the NCI in vitro screening with IC(50) values in the 0.1-5 nM range for most cell lines. Compounds 26 and 30 are highly active against L1210 leukemia in mice. Compound 26 is also active against B16BL6 melanoma in mice. Most importantly, 26 and 30 are not myelosuppressive at therapeutically effective doses. The mechanism of tumor cell death is through induction of apoptosis, and is accompanied by DNA fragmentation.     

Pseudolaric acid B induces mitotic catastrophe followed by apoptotic cell death in murine fibrosarcoma L929 cells

Pseudolaric acid B (PAB) is the primary biologically active compound isolated from the root bark of P. kaempferi Gordon. Previous studies have demonstrated that PAB arrests cells in G2/M phase in several cancer cell lines without significantly perturbing the G2/M transition-associated proteins. CylinB1, a marker for mitotic phase arrest, was up-regulated in cells treated with PAB. Therefore, we investigated whether PAB affects cell cycle progression at the mitotic phase. The mitotic index increased during a 24h treatment with PAB, suggesting that PAB arrested cell cycle progression at mitosis. In addition, after a prolonged mitotic arrest, the cells underwent mitotic catastrophe. After an extended treatment with PAB (longer than 24h), the protein levels of cylinB1 and cdc2 significantly decreased in both nuclear and cytosolic extracts. According to these results, we concluded that mitotic slippage could be due to the inactivation of the cylinB1-cdc2 complex resulting from prolonged treatment with PAB. The cells undergoing mitotic catastrophe died via apoptosis.     

Cell fate after mitotic arrest in different tumor cells is determined by the balance between slippage and apoptotic threshold

Microtubule poisons and other anti-mitotic drugs induce tumor death but the molecular events linking mitotic arrest to cell death are still not fully understood. We have analyzed cell fate after mitotic arrest produced by the microtubule-destabilizing drug vincristine in a panel of human tumor cell lines showing different response to vincristine. In Jurkat, RPMI 8226 and HeLa cells, apoptosis was triggered shortly after vincristine-induced mitotic arrest. However, A549 cells, which express a great amount of Bcl-x_L and undetectable amounts of Bak, underwent mitotic slippage prior to cell death. However, when Bcl-x_L gene was silenced in A549 cells, vincristine induced apoptosis during mitotic arrest. Another different behavior was found in MiaPaca2 cells, where vincristine caused death by mitotic catastrophe that switched to apoptosis when cyclin B1 degradation was prevented by proteasome inhibition. Overexpression of Bcl-x_L or silencing Bax and Bak expression delayed the onset of apoptosis in Jurkat and RPMI 8226 cells, enabling mitotic slippage and endoreduplication. In HeLa cells, overexpression of Bcl-x_L switched cell death from apoptosis to mitotic catastrophe. Mcl-1 offered limited protection to vincristine-induced cell death and Mcl-1 degradation was not essential for vincristine-induced death. All these results, taken together, indicate that the Bcl-x_L/Bak ratio and the ability to degrade cyclin B1 determine cell fate after mitotic arrest in the different tumor cell types.     

Pseudolaric Acid B–Induced Autophagy Contributes to Senescence via Enhancement of ROS Generation and Mitochondrial Dysfunction in Murine Fibrosarcoma L929 Cells

Pseudolaric acid B (PAB) is the primary biologically active compound isolated from the root bark of P. kaempferi Gordon. Our previous study demonstrated that PAB induced mitotic catastrophe in L929 cells and indicated that only a small percentage (12%) of the cells undergoing mitotic catastrophe displayed an apoptotic phenotype after PAB treatment for 72 h. In this study, we found that a minority of the cells undergoing mitotic catastrophe ended in apoptosis, and a majority of them entered a period of senescence. Further data confirmed that PAB induced autophagy, reactive oxygen species (ROS) generation, and mitochondrial dysfunction in L929 cells. Subsequently, we found that autophagy inhibitors significantly delayed the senescence process, indicating that autophagy facilitated senescence. Moreover, ROS scavenger significantly decreased the autophagic level and improved mitochondrial function. Additionally, autophagy inhibitors effectively reduced ROS levels and ameliorated mitochondrial function. In conclusion, autophagy promoted senescence via enhancement of ROS generation and mitochondrial dysfunction in PAB-treated L929 cells.     

放线菌素D不能抑制α-双炔失碳酯诱导的白血病K562细胞凋亡

目的：研究放线菌素Ｄ（ＡｃｔＤ）对α－双炔失碳酯（α－ａｎｏｒｄｒｉｎ，ＡＮＯ）诱导的人白血病Ｋ５６２细胞凋亡的影响。方法：用光学显微镜观察细胞形态学变化；用流式细胞仪、琼脂糖凝胶电泳分别检测ＤＮＡ含量和ＤＮＡ断裂。结果：ＡＮＯ５０μｍｏｌ·Ｌ－１处理人白血病Ｋ５６２细胞２４ｈ引起大约１０％Ｋ５６２细胞产生凋亡。同时加入ＲＮＡ合成抑制剂ＡｃｔＤ０．００５μｍｏｌ·Ｌ－１不能抑制ＡＮＯ诱导的凋亡，相反地，ＡｃｔＤ加强ＡＮＯ的这一作用，使凋亡细胞从１０％上升到２０％。ＡｃｔＤ０．５μｍｏｌ·Ｌ－１本身可诱导约３２％的Ｋ５６２细胞凋亡。Ｓ期细胞对ＡＮＯ诱导的凋亡较敏感，而ＡｃｔＤ则较易诱导Ｓ期和Ｇ２－Ｍ期细胞凋亡。结论：ＡＮＯ诱导的Ｋ５６２细胞凋亡不依赖于新的ＲＮＡ合成。     

Withaferin A modulates the Spindle Assembly Checkpoint by degradation of Mad2–Cdc20 complex in colorectal cancer cell lines

Withania somnifera L. Dunal (Ashwagandha) is used over centuries in the ayurvedic medicines in India. Withaferin A, a withanolide, is the major compound present in leaf extract of the plant which shows anticancer activity against leukemia, breast cancer and colorectal cancer. It arrests the ovarian cancer cells in the G2/M phase in dose dependent manner. In the current study we show the effect of Withaferin A on cell cycle regulation of colorectal cancer cell lines HCT116 and SW480 and its effect on cell fate. Treatment of these cells with this compound leads to apoptosis in a dose dependent manner. It causes the G2/M arrest in both the cell lines. We show that Withaferin A (WA) causes mitotic delay by blocking Spindle assembly checkpoint (SAC) function. Apoptosis induced by Withaferin A is associated with proteasomal degradation of Mad2 and Cdc20, an important constituent of the Spindle Checkpoint Complex. Further overexpression of Mad2 partially rescues the deleterious effect of WA by restoring proper anaphase initiation and keeping more number of cells viable. We hypothesize that Withaferin A kills cancer cells by delaying the mitotic exit followed by inducing chromosome instability.     

Mitotic catastrophe as a consequence of chemotherapy

According to a widespread model, anti-cancer chemotherapy involves the triggering of tumor cells to undergo apoptosis, so apoptosis-resistant cells would be recalcitrant to such therapy. However, in addition to apoptosis, which is mainly dependent on the activity of the tumor suppressor protein p53, cells can be eliminated following DNA damage by other mechanisms. Mitotic catastrophe, a form of cell death that results from abnormal mitosis, is one such mechanism. While the term mitotic catastrophe has been used to describe a type of cell death that occurs during mitosis, there is still no broadly accepted definition. Occasionally, mitotic catastrophe is used restrictively for abnormal mitosis leading to cell death, which can occur through necrosis or apoptosis, rather than cell death itself. Although different classes of cytotoxic agents induce mitotic catastrophe, the pathways of abnormal mitosis differ depending on the nature of the inducer and the status of cell-cycle checkpoints. Moreover, mitotic catastrophe can also develop because of aberrant re-entry of tumor cells into the cell cycle after prolonged growth arrest. Elucidation of the factors that regulate different aspects of treatment-induced mitotic catastrophe should assist in improving the efficacy of anti-cancer therapy, providing opportunities for the development of new drugs.     

2-(3-Methoxyphenyl)-5-methyl-1,8-naphthyridin-4(1H)-one (HKL-1) induces G2/M arrest and mitotic catastrophe in human leukemia HL-60 cells

2-(3-Methoxyphenyl)-5-methyl-1,8-naphthyridin-4(1H)-one (HKL-1), a 2-phenyl-1,8-naphthyridin-4-one (2-PN) derivative, was synthesized and evaluated as an effective antimitotic agent in our laboratory. However, the molecular mechanisms are uncertain. In this study, HKL-1 was demonstrated to induce multipolar spindles, sustain mitotic arrest and generate multinucleated cells, all of which indicate mitotic catastrophe, in human leukemia HL-60 cells. Western blotting showed that HKL-1 induces mitotic catastrophe in HL-60 cells through regulating mitotic phase-specific kinases (down-regulating CDK1, cyclin B1, CENP-E, and aurora B) and regulating the expression of Bcl-2 family proteins (down-regulating Bcl-2 and up-regulating Bax and Bak), followed by caspase-9/-3 cleavage. These findings suggest that HKL-1 appears to exert its cytotoxicity toward HL-60 cells in culture by inducing mitotic catastrophe.     

Antitumour imidazoacridone C-1311 induces cell death by mitotic catastrophe in human colon carcinoma cells

In this study, we investigated the cell death process induced by imidazoacridone C-1311 (Symadex) in HT-29 human colon carcinoma cells which have been shown to be preferentially sensitive to this compound in experimental tumour models both in vitro and in nude mice. Compound C-1311 at the EC(99) dose delayed progression of cells through the S phase which was followed by G2 arrest. At 48-96 h after drug exposure, an increasing fraction of cells rounded up and detached from the substratum which suggested the induction of cell death. This was confirmed by the induction of DNA fragmentation as revealed by pulse field electrophoresis and DNA strand breaks by the TUNEL assay. The dying cells had also mitotic features which were evidenced by various biochemical and morphological criteria such as activation of Cdk1 kinase, presence of the mitotic epitope MPM-2 and condensation of chromatin into mitotic chromosomes in drug-treated cells. These results show that C-1311 does not induce rapid apoptosis in HT-29 cells, instead drug exposure leads to prolonged G2 arrest followed by G2 to M transit and cell death during mitosis in the process of mitotic catastrophe.     

Induction of mitosis-mediated apoptosis by sodium arsenite in HeLa S3 cells

Arsenic has been used effectively as a chemotherapeutic drug for the treatment of acute promyelocytic leukemia patients. Numerous studies have demonstrated that arsenic induces apoptosis in various cell types. In the present study, we showed that approximately 35% of arsenite-treated HeLa S3 cells arrested in mitosis. After release from arsenite treatment, more than 80% of arsenite-arrested mitotic cells subsequently underwent apoptosis, as indicated by anachronistic nuclear envelope reformation, DNA ladder occurrence, chromatin condensation, and activation of caspases 3 and 9. In exploring how these cells entered apoptosis mechanistically, we found an inverse correlation between mitotic indexes and apoptotic frequencies. As shown by using Percoll density gradient fractionation and flow cytometric analysis, the mitosis-mediated apoptosis induced by arsenite was accompanied by delayed cyclin B degradation and altered mitotic exit. Furthermore, treatment of arsenite-arrested mitotic cells with staurosporine or 2-aminopurine resulted in a rapid degradation of cyclin B, moved these cells forward to interphase without cell division, and abrogated apoptosis. These results suggest that apoptosis occurs in arsenite-arrested mitotic cells that exit mitosis abnormally.     

Synthesis and evaluation of 4/5-hydroxy-2,3-diaryl(substituted)-cyclopent-2-en-1-ones as cis-restricted analogues of combretastatin A-4 as novel anticancer agents

A new series of 2,3-diaryl-4/5-hydroxy-cyclopent-2-en-1-one analogues replacing the cis double bond of combretastatin A-4 (CA-4) by 4/5-hydroxy cyclopentenone moieties was designed and synthesized. The analogues displayed potent cytotoxic activity (IC50<1 microg/mL) against a panel of human cancer cell lines and endothelial cells. The most potent analogues 11 and 42 belonging to the 5-hydroxy cyclopentenone class were further evaluated for their mechanism of action. Both of the analogues led to cell cycle arrest at G2/M phase and induced apoptosis in endothelial cells. Antitubulin property of 42 was superior to 11 and comparable to CA-4. The compound 42 had better aqueous solubility, metabolic stability, and pharmacokinetic profile than CA-4 and also demonstrated significant tumor regression in the human colon xenograft model. Our data suggests that cis-restricted analogues of CA-4 are a new class of molecules that have the potential to be developed as novel agents for the treatment of cancer.     

Genotoxicity, mitochondrial damage, and apoptosis in human lymphoblastoid cells exposed to peroxynitrite generated from SIN-1

SIN-1 (3-morpholinosydnonimine), the active metabolite of the vasodilator drug molsidomine, decomposes spontaneously in solution. In the presence of oxygen, NO* and O(2)(*-) are released, generating peroxynitrite, a potent oxidizing agent, at a constant rate over a 2 h period. We utilized this system to investigate mechanisms of peroxynitrite-induced cytotoxicity, genotoxicity, apoptosis, and mitochondrial damage in two human lymphoblastoid cell lines carrying either wild-type (TK6 cells) or mutant p53 (WTK-1 cells) genes. Treatment of TK6 cells with 5 mM SIN-1 for 1.5 h resulted in 28 +/- 6% survival 24 h later. Exposure in the presence of different radical scavengers significantly increased survival, as follows: cytochrome c, 96 +/- 3%; Tiron, 69 +/- 0%; SOD plus catalase, 83 +/- 5%; carboxy-PTIO, 87 +/- 3%; and uric acid, 87 +/- 2%. D-mannitol was ineffective in reducing lethality, as were SOD and catalase when added individually or in heat-inactivated form. Spontaneous as well as SIN-1-induced mutant fractions (MF) in both HPRT and TK genes were significantly higher in WTK-1 cells than in TK6 cells (p < 0.05-0.01). Exposure to 2.5 mM SIN-1 induced time-dependent apoptosis in TK6 cells, but not in WTK-1 cells. Mitochondrial membrane depolarization was also observed in both cell lines after SIN-1 treatment. Neutral comet assay demonstrated that SIN-1 treatment resulted in higher levels of DNA double-strand breaks in TK6 cells than in WTK-1 cells. Collectively, these data show that SIN-1 can be used as an effective peroxynitrite generator in cell culture experiments under these experimental conditions, in which it induced a greater apoptotic response but was less potent as a mutagen in TK6 cells compared with WTK-1 cells. Thus, p53 status was an important determinant of SIN-1 induced mutagenesis and apoptosis in these two human lymphoblastoid cell lines.     

舒尼替尼与Combretastatin A-4联合用药体外抑制人胃癌SGC-7901细胞增殖作用研究

目的 对舒尼替尼与Combretastatin A-4(CA-4)联合用药的抗肿瘤活性进行研究。方法 SRB法测定比较舒尼替尼、CA-4及联合用药对人胃癌SGC-7901细胞增殖抑制作用。PI染色法、DAPI染色法及Western blot法检测舒尼替尼联合应用CA-4诱导人胃癌SGC-7901细胞凋亡作用。结果 联用舒尼替尼和CA-4在体外能协同抑制人胃癌SGC-7901细胞的增殖。舒尼替尼联用CA-4能够诱导SGC-7901肿瘤细胞凋亡,并且伴随着caspase-3的激活和PARP的裂解。结论 舒尼替尼与CA-4联合用药在体外能协同诱导人胃癌SGC-7901肿瘤细胞增殖。