Cancer chemo-endocrine therapy and its cell biological basis

The aim of our cell kinetic studies is to better understand the effects of chemo-endocrine therapy at the cell biological and molecular level. Cancer cell growth is characterized by uncontrolled proliferation, resulting in DNA distribution pattern in which, at any time, more cells are not G1 phase but in S, G2 and M phase of a shortened cycle. In a recent progress, flow cytometry (FCM) has become a powerful tool for the quantitative analysis of cell cycle parameters by measuring nuclear DNA content in large cell population with high speed. With the aid of FCM in earlier work about 60-80% of ovarian cancers were found to contain aneuploid cells. Now, multi-parameter FCM linked to a computer is available to measure fluorescent intensities not only no base total DNA (Propidium iodide) but also A-T (Hoechst 33342) and G-C (Mithramycin) base pairs in solid cancer nuclei. Since cisplatinum (CDDP) is the most important drug in the treatment of ovarian cancer, we have studied the relationship of CDDP cytotoxicity, pertubations cell cycle kinetis and DNA damage in ovarian adenocarcinoma cells in vitro & in vivo. We employed both CDDP sensitive cell line (KFt) and resistant cell line (KFr) derived from human serous cystoadenocarcinoma of the ovary by Kikuchi et al (JNCI 1986). Comparing cell kinetic pertubations of experimental cells demonstrates a decrease in G1 phase cells concomitant increase in S phase cells. The KFr cells had distinctly a shorter S-phase block up to 24 hrs not A-T but G-C preference in a quick response followed repairing of DNA damage to 48 hrs. However, some fractions of CDDP resistant cell population showed a later onset of G2, M phase accumulation. Comparison with the increase in early S phase cells of KFr in detailed analysis suggests only those damaged cells that are not killed immediately may proceed to G1 phase and start into DNA synthesis in S phase. Measurement of labeling index (L. I.) with Bromodeoxyuridine (BrdU) support our interpretation of differences between sensitivity and resistance to anti-cancer drug. Additionally, we discuss a targeting chemotherapy by coupling cytotoxic drugs with estrogen based on increasing DNA damage into apoptosis and interfares with DNA repair process.     

Experimental and clinical evaluation by flow cytometry for the mechanism of combination therapy (cisplatin and peplomycin).

Pharmacologic effects of cisplatin (CDDP) and peplomycin (PEP) on tumor cell kinetics were studied both in vitro and in vivo with the aid of flow cytometry (FCM). Double staining with propidium iodide (PI) and fluorescein isothiocyanate (FITC)-labeled bromodeoxyuridine (BrdU) was used to analyze the cell cycle, and the number of viable cells was determined with fluorescein diacetate (FDA). Effects of combining the 2 agents were also studied to establish the most effective method of combination therapy. Furthermore, these agents were tried clinically on the basis of experimental results. Results showed that CDDP exerted its action at the S and G2M phases in the cell cycle and PEP at the G2M phase. Among the combination regimens in the experiments with CDDP, PEP, and CDDP + PEP as analyzed by FCM, the strongest block on the G2M phase was shown in the one at a 2-day interval, resulting in the most effective killing of the tumor cells. Clinical trial of the combination therapy showed the same results as the in vitro experiment; the therapy proved useful for improving the patient's clinical condition and the results obtained with CT imaging and pathology.     

EBNA1 may prolong G(2)/M phase and sensitize HER2/neu-overexpressing ovarian cancer cells to both topoisomerase II-targeting and paclitaxel drugs

We have shown previously that the Epstein-Barr virus nuclear antigen-1 (EBNA1) can act as a transforming suppressor in the HER2/neu-overexpressing ovarian cancer cells. In the present study, by using flow cytometric analysis, we demonstrate that EBNA1 could prolong G(2)/M phase and sensitize to Taxol-induced apoptosis in the EBNA1-expressing ovarian cancer cell stable transfectants. In addition, EBNA1 could also significantly increase topoisomerase IIalpha protein expression, indicating that the up-regulation of topoisomerase IIalpha may be one of the mechanisms by which EBNA1 enhances the sensitivity of ovarian cancer cells to topoisomerase II-targeting anticancer drugs, such as VP-16 and Adriamycin. These data suggest that EBNA1 not only prolongs cell cycle at G(2)/M phase and up-regulates topoisomerase IIalpha expression in HER2/neu-overexpressing ovarian cancer cells, but also increases cellular apoptosis through sensitization of cancer cells to topoisomerase II-directing anticancer drugs.     

Changes in cell cycle and RNA content in cultured cancer cells treated with cis-diamminedichloroplatinum (II)

Recent reports have shown that CDDP interacts with RNA and protein as well as DNA. We studied the alteration of cell cycle, cellular RNA content and the effect of nucleic acid metabolism on cultured cancer cells after treatment with CDDP by flow cytometry and 3H incorporation assay. The alteration of cell cycle was found to be accumulation of cells in after delay S phase in cytostatic concentrations, CDDP inhibited 3H-TdR uptake markedly at this time and 3H-UR uptake earlier. Increase in RNA content accompanied accumulation of cells in G2M phase. This increase was not a specific phenomenon caused by CDDP, because increase in RNA content was also induced by other inhibitors of DNA synthesis. It is more likely that the direct alteration of cell cycle and cellular RNA content due to action of DNA-combined CDDP rather than that of RNA-combined CDDP.     

Increased mitochondrial uptake of rhodamine 123 by CDDP treatment.

Rhodamine 123 (R 123) is a positively charged dye at physiological pH that accumulates specifically in the mitochondria of living cells without cytotoxic effect. In the present study, the uptake of R 123 by EL-4 lymphoma cells in culture with anticancer agents was measured by flow cytometry. Changes in R 123 uptake during the cultivation period were compared with cell distribution at different phases of the cell cycle. According to the increase in the proportion of S phase cells, mitochondrial synthesis increased, giving rise to a maximal fluorescence intensity of about 1.3-fold. Synchronous cultures showed the same relationship between increased mitochondrial uptake of R 123 and the S phase fraction as was observed in normal cultures. After treatment with 10(-3) M 5-fluorouracil (5-FU) for 1 h, EL-4 cells showed an increased binding of R 123 per cell followed by an accumulation of early S phase cells transiently. However, uptake of R 123 decreased 24 h later. On the contrary, after treatment with 10 micrograms/ml of cis-diamminedichloroplatinum (CDDP), a G2 + M block was observed from 12 h of reseeding and accumulation of the G2 + M cells continued. In this case, high uptake of R 123 continued during the observation period. From these results, mitochondrial synthesis seemed to increase according to the increment in proportion of S phase when the acceleration of the cell cycle turnover was augmented or the cycle was blocked in S phase by 5-FU. CDDP inhibited the cell division at G2 + M phase and caused increased R 123 fluorescence per cell. The stainability of R 123 may indicate the activity of cell division and may be a good way of evaluating the efficacy of antitumor drugs on the cells.     

Phosphorylation of mixed lineage kinase MLK3 by cyclin-dependent kinases CDK1 and CDK2 controls ovarian cancer cell division

Mixed lineage kinase 3 (MLK3) is a serine/threonine mitogen-activated protein kinase kinase kinase that promotes the activation of multiple mitogen-activated protein kinase pathways and is required for invasion and proliferation of ovarian cancer cells. Inhibition of MLK activity causes G2/M arrest in HeLa cells; however, the regulation of MLK3 during ovarian cancer cell cycle progression is not known. Here, we found that MLK3 is phosphorylated in mitosis and that inhibition of cyclin-dependent kinase 1 (CDK1) prevented MLK3 phosphorylation. In addition, we observed that c-Jun N-terminal kinase, a downstream target of MLK3 and a direct target of MKK4 (SEK1), was activated in G2 phase when CDK2 activity is increased and then inactivated at the beginning of mitosis concurrent with the increase in CDK1 and MLK3 phosphorylation. Using in vitro kinase assays and phosphomutants, we determined that CDK1 phosphorylates MLK3 on Ser548 and decreases MLK3 activity during mitosis, whereas CDK2 phosphorylates MLK3 on Ser770 and increases MLK3 activity during G1/S and G2 phases. We also found that MLK3 inhibition causes a reduction in cell proliferation and a cell cycle arrest in ovarian cancer cells, suggesting that MLK3 is required for ovarian cancer cell cycle progression. Taken together, our results suggest that phosphorylation of MLK3 by CDK1 and CDK2 is important for the regulation of MLK3 and c-Jun N-terminal kinase activities during G1/S, G2, and M phases in ovarian cancer cell division.     

The anti-tumor effect of cross-reacting material 197, an inhibitor of heparin-binding EGF-like growth factor, in human resistant ovarian cancer

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a promising target for ovarian cancer therapy. Cross-reacting material 197 (CRM197), a specific HB-EGF inhibitor, has been proven to represent possible chemotherapeutic agent for ovarian cancer. However, the effect of CRM197 on the resistant ovarian carcinoma cells has not been sufficiently elucidated. Here, we found that HB-EGF was over-expressed in a paclitaxel-resistant human ovarian carcinoma cell line (A2780/Taxol) and a cisplatin-resistant cell line (A2780/CDDP), as well as the xenograft mouse tissue samples with these cells. To investigate the possible significance of the HB-EGF over-expression in A2780/Taxol and A2780/CDDP cells, we inhibited HB-EGF expression by CRM197 to investigate the effect of CRM197 treatment on these cells. We observed that CRM197 significantly induced anti-proliferative activity in a dose-dependent manner with the cell-cycle arrest at the G0/G1 phase and enhanced apoptosis in A2780/Taxol and A2780/CDDP cells. The sensitive ovarian carcinoma parental cell line (A2780), A2780/Taxol and A2780/CDDP cells formed tumors in nude mice, and enhanced tumorigenicity was observed in drug-resistant tumors. Furthermore, we observed that CRM197 significantly suppressed the growth of drug-resistant ovarian cancer xenografts in vivo (p<0.001). These results suggest that CRM197 as an HB-EGF-targeted agent has potent anti-tumor activity in paclitaxel- and cisplatin-resistant ovarian cancer which over-express HB-EGF.     

卡铂通过抑制ERK1/2通路诱导人卵巢癌细胞S和G0/G1期阻滞

卡铂(carboplatin, CBP)是一种抗肿瘤活性较强的化疗药物, 通过诱导细胞周期阻滞抑制肿瘤细胞生长, 但其诱导细胞周期阻滞的报告不甚一致. 本研究探索卡铂对卵巢癌HO-8910细胞生长及细胞周期进程的影响. MTS结果显示, 卡铂以浓度和时间依赖方式抑制卵巢癌HO-8910细胞生长, 联合使用ERK1/2通路抑制剂PD98059可使卡铂抗卵巢癌细胞增殖作用增强. 采用Giemsa染色法观察到, 卡铂与PD98059单用或联用均能致卵巢癌细胞发生明显的形态学变化. 流式细胞术检测细胞周期发现, 随卡铂浓度的增高, S期阻滞作用增强; 抑制ERK1/2通路可拮抗卡铂对HO-8910细胞S期阻滞作用, 增加G1期阻滞作用, 而对G2/M期细胞影响不明显. Western印迹结果显示, 随卡铂浓度的增高, p-ERK1/2、Cdc2(Y15)和p Cdc2(T161)的表达逐渐升高, Cyclin E1和Cyclin B1的表达逐渐降低; 抑制ERK1/2通路可将卡铂上调,p-ERK1/2和p-Cdc2(T161)的作用反转为下调作用, 上调Cdc2(Y15)的表达受阻, 抑制Cyclin B1的下调作用, 促进Cyclin E1的下调作用. 本研究结果提示, 卡铂通过抑制ERK1/2激活, 诱导人卵巢癌HO-8910细胞S和G1期阻滞, 抑制卵巢癌细胞生长.     

顺铂靶向食管癌细胞周期Cx43表达的研究

目的:为研究顺铂治疗食管鳞癌细胞的靶向作用。方法:本研究使用流式细胞技术双变量分析检测顺铂对食管癌细胞周期进程和癌细胞周期的连接蛋白43(connexin 43,Cx43)表达的影响。结果:顺铂对食管鳞癌细胞周期的影响主要作用于S期的DNA复制,细胞阻滞于S期,G2/M期减少。顺铂诱导食管鳞癌细胞周期S和G2/M期的Cx43表达的大幅度改变。低浓度顺铂(由0~2μmol/L),Cx43表达增强;顺铂渐高浓度(2~12μmol/L),细胞Cx43表达由强逐渐变弱,特别是G2/M期细胞的Cx43表达活跃,易受顺铂影响。结论:我们的研究表明以顺铂处理食管鳞癌细胞,癌细胞周期的S期和G2/M期的Cx43表达与S期的DNA复制一样可作为的潜在治疗靶标。顺铂靶向作用细胞周期S和/或G2/M期细胞的特性可能减少或避免对非分裂细胞的影响。     

Blocking the NOTCH pathway can inhibit the growth of CD133-positive A549 cells and sensitize to chemotherapy

Cancer stem cells (CSCs) are believed to play an important role in tumor growth and recurrence. These cells exhibit self-renewal and proliferation properties. CSCs also exhibit significant drug resistance compared with normal tumor cells. Finding new treatments that target CSCs could significantly enhance the effect of chemotherapy and improve patient survival. Notch signaling is known to regulate the development of the lungs by controlling the cell-fate determination of normal stem cells. In this study, we isolated CSCs from the human lung adenocarcinoma cell line A549. CD133 was used as a stem cell marker for fluorescence-activated cell sorting (FACS). We compared the expression of Notch signaling in both CD133+ and CD133− cells and blocked Notch signaling using the γ-secretase inhibitor DAPT (GSI-IX). The effect of combining GSI and cisplatin (CDDP) was also examined in these two types of cells. We observed that both CD133+ and CD133− cells proliferated at similar rates, but the cells exhibited distinctive differences in cell cycle progression. Few CD133+ cells were observed in the G₂/M phase, and there were half as many cells in S phase compared with the CD133− cells. Furthermore, CD133+ cells exhibited significant resistance to chemotherapy when treated with CDDP. The expression of Notch signaling pathway members, such as Notch1, Notch2 and Hes1, was lower in CD133+ cells. GSI slightly inhibited the proliferation of both cell types and exhibited little effect on the cell cycle. The inhibitory effects of DPP on these two types of cells were enhanced when combined with GSI. Interestingly, this effect was especially significant in CD133+ cells, suggesting that Notch pathway blockade may be a useful CSC-targeted therapy in lung cancer.     

Impaired hydrolysis of cisplatin derivatives to aquated species prevents energy-dependent uptake in GLC4 cells resistant to cisplatin

It has been widely stated that cisplatin enters cells by passive diffusion, despite some reports supporting a carrier-mediated mechanism. We have determined the rate of uptake of carboplatin (CBDCA), of cisplatin (CDDP) and of aquated forms, at different values of the extracellular pH, in the small lung-cancer cells GLC4 and GLC4/CDDP, cells resistant to CDDP. The rate of CDDP uptake is about 2-fold lower in resistant cells than in sensitive ones; in ATP-depleted cells this rate is about the same for both cell lines. The rate of CBDCA uptake is about 10-fold lower than that of CDDP and is the same in both cell lines independently of the ATP status of the cells. On the other hand, the rate of uptake of the aquated form of CDDP is approximately 40-fold higher than that of CDDP and is the same in both cell lines, but decreases dramatically in ATP-depleted cells. The plot of the initial rate of uptake of the aquated species as a function of its extracellular concentration shows a tendency to be saturable with k(m)=1.9 mM. In conclusion, our data show that, in sensitive GLC4 cells, passive diffusion of CDDP, probably in its neutral dichloro form, and active uptake of the aquated form contribute to the platinum uptake. The active transport of CDDP involves at least two steps: (1). the hydrolysis of the dichloro species in a deficient Cl(-) space at the level of the plasma membrane, which is the limiting step, and (2). the active transport of the aquated species. In resistant cells, step (1). should be deficient whereas step (2). is the same as in sensitive cells. For CBDCA this mechanism holds; however, step (1). is so low that the active transport does not contribute to the uptake of CBDCA by cells.     

Cytokinetic effects of cisplatin on cisplatin-resistant ovarian cancer cell lines]

Cytokinetic effects of cisplatin on human ovarian cancer cell lines with natural cisplatin-resistance was examined by means of flow cytometry. These ovarian cancer cell lines derived from patients with clear cell carcinoma and serous cystadenocarcinoma were established and designated "KK" and "MH", respectively. Both KK and MH cells have shown resistance to cisplatin and IC50 of them were 0.95 microM and 3.28 microM, respectively. Cisplatin inhibited cell cycle progression at G2 +M phase up to IC50 of each cell from the analysis of cell cycle. Similar results had been obtained in the case of "KF" cell which was sensitive to cisplatin. Further studies of these cells should be performed to elucidate the mechanism of cisplatin resistance.     

Synthesis and anti-cancer activity of chalcone linked imidazolones

A series of novel chalcone linked imidazolones were prepared and evaluated for their anti-cancer activity against a panel of 53 human tumour cell lines derived from nine different cancer types: leukemia, lung, colon, CNS, melanoma, ovarian, renal, prostate and breast. Some of these hybrids (6, 7 and 8) showed good anti-cancer activity with GI₅₀ values ranging from 1.26 to 13.9 μM. When breast carcinoma cells (MCF-7) were treated with 10 μM concentration of compounds TMAC, CA-4, 6 and 8 cell cycle arrest was observed in G2/M phase. Surprisingly, the increased concentration of the same compound to 30 μM caused accumulation of cells in G0/G1 phase of the cell cycle.     

1,25-Dihydroxyvitamin D3 and the regulation of human cancer cell replication

Several human and animal cancer cell lines have been shown to possess specific high affinity receptors for 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). The replication of several of these cell types has also been shown to be regulated by this hormone, both in vitro and in vivo. To further understand the mechanisms of these actions, we have examined cancer cells in vitro and in vivo. The in vitro studies extend our previous reports on the treatment of human breast cancer cells (T 47D) with 10(-9) to 10(-6) M 1,25-(OH)2D3, which resulted in a dose- and time-dependent decrease in cell numbers over 6 days. Treatment with 10(-8) M 1,25-(OH)2D3, which reduced cell numbers to approximately one half of those found in control cultures at 6 days, was associated with a doubling of the proportion of cells in the G2 + M phase of the cell cycle and was accompanied by a significant decline in the proportion of G0/G1 cells. At higher concentrations there was a significant decline in S phase cells with accumulation of cells in both G0/G1 and G2 + M phases. The antiestrogen, tamoxifen, at a concentration which caused similar effects on cell number, resulted in proportional decreases in both S and G2 + M phase cells and accumulation of G0/G1 cells. The effects of 1,25-(OH)2D3 on T 47D cell proliferation were associated with time- and concentration-dependent reductions in epidermal growth factor receptor levels to a minimum level of about half that seen in control cultures. The in vivo experiments extend our previous studies, which demonstrated marked inhibition of the growth of human cancer xenografts in immunosuppressed mice by 1,25-(OH)2D3. Xenograft growth was inhibited with 1,25-(OH)2D3 (0.1 microgram ip three times per week) but growth was rapidly restored when the 1,25-(OH)2D3 was withdrawn. Thus, there are clear-cut time- and dose-dependent, yet reversible, effects of 1,25-(OH)2D3 on the replication of human cancer cells in vitro and in vivo, which are possibly mediated through changes in growth factor receptor levels. Further study of these effects may advance understanding of the hormonal control of cellular replication in human cancers.     

Synchronization of the cell cycle using Lovastatin

Synchronization by Lovastatin arrests many cell types reversibly in the G1 phase of the cell cycle. Here we show that Lovastatin (10 µM) mediates cell cycle arrest in human breast cancer cells, MCF-7 and MDA-MB-231, where 85% of cells accumulate in the G1 phase of the cell cycle. Addition of mevalonate (at 100X the Lovastatin concentration) releases the cells from the G1 arrest and allows for synchronous entry into late G1, S and G2/M phases of the cell cycle. The expressions of different cyclins as a marker for different phases of the cell cycle are detected by western blot analysis and indicative of synchronous transition into each of cell cycle phases following the initial G1 arrest. Due to its level of synchrony and high yield of synchronous populations of cells, Lovastatin method of cell synchronization can be used for examining gene expression patterns in a variety of different cell lines.     

The difference in murine CDC2 kinase activity between cytoplasmic and nuclear fractions during the cell cycle

The mouse analog of yeast CDC2+ kinase was detected in the cytoplasmic and nuclear fractions of cultured mouse FM3A cells. Its activity in the nuclear fraction increased in the G2/M phase became seven times higher than that in the G1/S phase, while the activity in the cytoplasmic fraction remained was almost constant from the G1/S to G1 phases. The activity in the cytoplasmic fraction was similar to that in the nuclear fraction in the G2/M phase. The amount of the enzyme remained almost constant during the cell cycle in both the nuclear and cytoplasmic fractions. These findings suggest that the cytoplasmic enzyme might play an independent role in the cell cycle.     

Synthesis of a new 4-aza-2,3-didehydropodophyllotoxin analogues as potent cytotoxic and antimitotic agents

A series of novel conjugates of 4-aza-2,3-didehydropodophyllotoxins (11a-w) were synthesized by a straightforward one-step multicomponent synthesis that demonstrated cytotoxicity against five human cancer cell lines (breast, oral, colon, lung and ovarian). All the twenty three compounds (11a-w) have been examined for the inhibition of tubulin polymerization. Among these compounds, 11a, 11k and 11p exhibited inhibition of polymerization tubulin comparable to podophyllotoxin apart from disruption of microtubule organization within the cells. Flow cytometric analysis showed that these compounds (11a, 11k and 11p) arrested the cell cycle in the G2/M phase of cell cycle leading to caspase-3 dependent apoptotic cell death.     

Protein expression in relation to the cell cycle of exponentially growing human prostatic epithelial cells

This report concerns the study of the relationship between protein expression and the cell cycle in exponentially proliferating benign and malignant human prostate epithelial cells in short-term cultures. Multiparameter flow cytometric measurements were performed to correlate the expression of prostate-specific acid phosphatase, epithelial membrane antigen and epitectin with cell cycle progression. The expression of the three proteins was heterogeneous in G1 cells. The early post-mitotic cells exhibited the lowest levels when compared with late G1 cells, wherein the expression was many times greater. There was no further increase as the cells progressed through S and G2 + M. These findings, corroborating prior observations in other systems, suggest the possibility that the levels of the proteins studied increase during the G1 phase of the cell cycle and drop during or immediately after cytokinesis. As an alternate explanation, the heterogeneity of protein expression characteristic of G1 cells may be due, at least in part, to an asymmetric apportionment of cell constituents at mitosis.     

Identification and expression profiles of genes and protens in SMMC-7721 cells

In the study presented here, we first evaluated effect of CDDP on liver cancer cells SMMC-7721 apoptosis and motility capacity. Then, we evaluate inhibitory effect of CDDP on tumour growth and its possible molecular mechanism in liver cancer mice model. Results showed that the apoptosis rate of cells decreased with increasing CDDP. Analysis of the effect of the CDDP on cell cycle was performed by flow cytometry and results show a dose-dependent increase in the percentage of cells in the S-phase of the cell cycle, with a decrease in the percentage of cells in the G1 and G2/M phases. CDDP did not close the wound even after 48 h, as opposed to untreated cells (0 mg/l). Similarly, the migratory and invasion capacity of SMMC-7721 cells was also reduced after treatment with CDDP, as evaluated by a transwell assay. Animal experiment indicated that CDDP administration could increase blood WBC, total protein, albumin and A/G, decrease blood alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase levels in hepatocellular carcinomas mice. Immunohistochemistry analysis showed that positive expression of Fas and Bax proteins in the medicine-treated (II, III) group was significantly higher, whereas the expression of NF-κB, P53, Bcl-2 proteins was significantly lower than those of the control group. Gene expression analysis using Real time PCR methods revealed a significant up-regulation in the expression levels of Bax mRNA in the medicne-treated (II, III) group when compared to untreated control. In contrast, CDDP-treated group showed a significant down regulation in the expression levels of Bcl-2 mRNA as compared to untreated control group. These results are in agreement with immunohistochemistry data. Our observations indicate that CDDP has damaged effects on liver tumour cells SMMC-7721 including apoptosis, motility and cell cycle under in vitro. CDDP can enhance pro-apoptosis gene Fas, Bax expression, decrease anti-apoptosis genes Bcl-2 expression, and mutant genes P53, NF-κB proteins expression.