| Background&Objective:The Third National Mortality Retrospective Sampling Survey showed that there were three characteristics of malignant tumor change in our country.1. The mortality rate and incidence rate of esophageal cancer, gastric cancer, cervical cancer, and nasopharyngeal carcinoma had dropped noticeably, especially cervical cancer.2. The mortality rate and incidence rate of lung cancer, liver cancer, colorectal cancer, breast cancer, bladder cancer, of which the occurrence were related to environment and lifestyle, had a clear upward trend, especially lung cancer and breast cancer. The incidence in lung cancer and breast cancer had raised at465%and96%in the past30years. Lung cancer had replaced liver cancer as the first leading cause of cancer death in urban and rural areas (About22.7%of fatalities of death in malignant tumor patients was caused by lung cancer).The incidence and mortality of lung cancer in all kinds of malignant tumors is in the first place in the world. According to the statistical report of the38th Annual Meeting of the American Society of Clinical Oncology, lung cancer each year causes1.4million deaths worldwide, and has an upward trend.Lung cancer in early stage has no obvious symptoms, especially non-small cell lung cancer (NSCLC) accounted for about80%of the total number of lung cancer. About65%-80%of patients diagnosed lung cancer were of advanced stage and had cancer metastasis thus had no opportunities of surgical operation and radiotherapy. So, the chemotherapy is irreplaceable in the treatment of lung cancer. Unfortunately, there was lacking of the long-term effectively chemotherapeutics for lung cancer. Many anti-cancer drugs are not suitable for the majority of lung cancer patients as its limited applicable indication, even emerging molecular targeted drugs with its strong specificity and low toxicity. International Cancer Organization recommends cisplatin-based combination chemotherapy as the standard chemotherapeutic scheme for advanced non-small cell lung cancer patients based on long-term clinical studies. In clinical practice, combination therapy generally used cisplatin combined with other chemotherapeutic agent, such as etoposide, doxorubicin and so on. A number of randomized phase III clinical trials and important collaborative organization studies and meta-analysis showed that the efficacy of different platinum-containing program is approximate and there is no platinum-free program with better overall efficacy than platinum-containing program. But the extensive application of platinum inevitably caused drug-resistance of tumor cells, thus the effect of chemotherapy is significantly reduced. This is a major problem facing the lung cancer treatment.The main cause of tumors refractory is development of multidrug resistance, in addition to the side effects of the chemotherapy drugs. According to research data released by the U.S. National Cancer Institute, death from inherent multidrug resistance accounted for about61%in the annual cancer patient death, and death from acquired resistance accounted for about33%. It means that more than90%of cancer patients with chemotherapy failed died for drug resistance. Not only the traditional chemotherapy drugs, but also the small molecule targeted therapies considered as landmark, such tyrosine kinase signal transduction pathway inhibitor, have widespread resistance. The drug-resistance of tumor is a major threat to effective treatment and reversing the cancer chemotherapy resistance has become an urgent task facing the medical profession. At the same time, looking for the platinum-resistance reversal agents is the key of the clinical treatment of lung cancer. Unfortunately, the formation mechanism of tumor cell resistance to platinum is unclear. Some studies showed that development of cisplatin resistance in tumor cells was related to the reduction of cellular platinum content, but other experiment data showed that it was not correct. So, there is no appropriate platinum-resistance reversal agents used in clinical treatment.Recently, the results of researches on histone deacetylase inhibitors (HDACi) are encouragingly. Targeting on histone deacetylase (HDAC), they could effectively inhibit tumor cell proliferation and induce cell apoptosis. At the same time, they could reverse multidrug resistance of tumor cells. For example, they could resensitize ovarian carcinoma cells and colon cancer cells to cisplatin and5-FU. These data suggested that HDACi had great potential value for drug exploitation.Histone has five main sub-types which demonstrate a high degree of structural similarity. Acetylation of the N-terminal tails of the core histones undergo post-translational modification may neutralizes positively charge, thus weaken interactions with the DNA, resulting in an ’open’ chromatin conformation and facilitating access for transcriptional regulators. The level of histone acetylation is determined by the activities of histone acetyltransferases (HAT) and histone deacetylases (HDAC). The dynamic balance between HAT and HDAC regulates chromosome structure and gene expression. Plenty of studies points to HDAC as a key enzyme in the regulation of gene expression, its abnormality play an important regulatory role in the occurrence and development of tumors. Therefore, histone deacetylase (HDAC) is considered as one of the most potential targets in anti-cancer treatment. SAHA was approved by the US FDA in October2006as a new class of anticancer agents that inhibited histone deacetylases. Present there are more than a dozen compounds undergoing clinical trials.In summary, we believe that the histone deacetylase inhibitor (HDACi) is expected to become the new multidrug-resistance reversal agent. In this experiment, we adopted trichostatin A (TSA) and valproic acid (VPA) as representatives of HDACi to evaluate the reversal effect on cisplatin-resistance in non-small cell lung cancer A549/DDP cells, and to explore its mechanisms. Methods&Content:The study is divided into two parts to carry out:1. To evaluate the reversal effect of HDACi on cisplatin-resistance in NSCLC A549/DDP cell:MTT test was used to determine the resistance index on cisplatin between cisplatin-sensitive A549cells and cisplatin-resistance A549/DDP cells and to evaluate the reversal effect of TSA or VPA combined with DDP in A549/DDP cells; Flow cytometer was used to detect the impact of a combined treatment of the TSA and DDP on cell apoptosis and cell cycle distribution; β-galactosidase staining was used to observe the aging induction of HDACi combined with DDP; Gene chip technology was used to analyze the regulation effect of HDACi combined with DDP on cell cycle related gene expression and through further adoption of western blot to confirm the impact of drugs on related protein expression.2. According to the results of the first part of the study, we plan to explore the HDACi mechanism of cellular senescence induction and cisplatin-resistance reversal effect from the point of cellular senescence:Western blot was used to detect the regulatory effect of HDACi on INK4a/ARF/RB pathway and Polycomb Group (PcG) protein.Results:1. IC50of A549and A549/DDP cells on DDP was6μmol/L and35μmol/L respectively, A549/DDP cell line was markedly resistant to cisplatin, the value of resistant fold for A549/DDP to cisplatin was5.8fold; TSA and VPA applied separately could markly inhibit the cell growth by a dose-dependent manner, IC50of A549/DDP was193nmol/L on TSA and7mmol/L on VPA.2. The cell growth inhibitory rate of TSA or VPA combined with DDP group was significantly higher than single group.3. Cell cycle experiment results showed that TSA combined with DDP significantly increased cell number of G2/M state in A549/DDP at48h compared with TSA group and DDP group, the difference was statistically significant (p<0.01).4. The morphological changes of A549/DDP cells caused by TSA combined with DDP under the microscope:cells becomed big and flat, particulate matter increased. Stained by β-galactosidase, many cells were colored blue.5. Both the gene chip and western blot results showed that TSA combined with DDP significantly increased the expression of p21, western blot showed that TSA could significantly up-regulate the expression of p21.6. TSA combined with DDP could significantly up-regulate expression level of p16,p14in A549/DDP.7. Compared with A549, the Polycomb Group (PcG) proteins (EZH2, SUZ12, RBAP46/48, EED, CBX7, BMI-1, and RING1B) and p-RB protein were overexpressed in A54P/DDP.8. DDP could up-regulate the expression of PcGs and p-RB in both A549and A549/DDP.9. TSA decreased proteins expression of PcGs, p-RB and RB in A549/DDP.Conclusion:1. TSA, VPA markedly enhanced the cytotoxicity of DDP on A549/DDP and had the effect of reversing cancer cisplatin-resistance.2. TSA combined with DDP could arrest cell cycle in G2/M phase to induce cellular senescence of A549/DDP.3. TSA increased the expression of p21, p16, p14and decreased the expression of RB and p-RB. These data suggested that the drugs induce the cellular senescence through the INK4a/ARF/RB pathways.4. DDP could up-regulate the expression of PcGs and PcGs were overexpressed in A549/DDP compared with A549. It suggested that PcG proteins are likely to participate in the cisplatin resistance formation and maintenance process of A549/DDP. TSA significantly inhibited the expression of PcGs. It suggested that reversal effect of TSA on cisplatin-resistance of A549/DDP is relevant to PcG proteins. |
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