Abstract Objective To detect the expression of resistance genes in human gastric carcinoma and selected according to test results sensitive chemotherapy explore the significance of the resistance gene detection of gastric cancer chemotherapy. 80 cases confirmed by endoscopy and pathology of gastric cancer patients, according to the number pre-adaptation group and the non-fit group 2 groups were randomly divided into 40. Immunohistochemistry was used to detect all cases of gastric cancer tissues of P gp, GST π, Topo Ⅱ expression. According to the test results, the adapter patients selection the corresponding the sensitive drugs adaptation chemotherapy, non-adaptation the group unified chosen FOLFOX4 chemotherapy. Results P gp, GST π, Topo Ⅱ expression rates were 51.5%, 57.5%, 46.3%; P gp, Topo Ⅱ expression of tumor differentiation degree of differentiation by expression of P gp High higher than other differentiation of non-high school, Topo Ⅱ the high differentiated by expression rate is lower than those of other non-high school differentiation; unrelated GST π expression and degree of differentiation, moderately differentiated differentiation of non-high school was no significant difference. Adapter group, chemotherapy effective rate of 57.5%, the effective rate of 35% of the non-fit group, the difference was statistically significant (P <0.05). Conclusion of P gp, GST π, Topo II resistance mechanism of the same for different patients, according to the resistance gene test results, the implementation of scientific, drug adaptation of individual chemotherapy for the treatment of gastric important guidance significance, can enhance the effect of chemotherapy.
Key words gastric cancer; P glycoprotein; glutathione S-transferase; topoisomerase II; multidrug resistance; chemotherapy
Significance of Testing Multiple Drug Resistance Gene of Gastric Carcinoma in Guiding Clinical Chemotherapy
JIA Chang he1, KANG Yi1, WANG Wen yu2, REN Ying3
1. Department of Gastroenterology, He’nan Provincial People’s Hospital, Zhengzhou 450003, China, 2. Department of Oncology, 3. Department of PathologyAbstract: Objective To test the expressions of multiple drug resistance (MDR) gene of human gastric carcinoma, and investigate its significance in guiding gastric carcinoma clinical chemotherapy by choosing appropriate remedy according to examination result.Methods Eighty gastric cancer patients diagnosed by endoscopy and pathology were random divided into two groups (fit group and non fit group), each included 40 patients. The expression of P gp, GST π, Topo Ⅱ was tested in 80 gastric cancer specimen using immunohistochemistry. The fit group patients were treated by choosing sensitive chemical remedy fitting for gene examination result.The non fit group patients were treated with the experiential chemotherapeutic scheme of FOLFOX4. Results Positive expression rates of P gp, GST π, Topo Ⅱ in 80 cases of gastric cancer were 51.5%, 57.5%, 46.3%, respectively. There were some correlations between the positive expression of P gp, Topo Ⅱ and the degree of tumor differentiation, which was higher in well differentiated tumor than that in poor differentiated one for P gp, but it was reverse for Topo Ⅱ. There were no correlations between the positive expression of GST π and the degree of tumor differentiation. The response rate was 57.5% in the fit group, which was 35% in the non fit group. There were significant different between the two groups (P <0.05). Conclusion The mechanisms of drug resistance of P gp, GST π, Topo Ⅱ in gastric cancer are different.It is of significance for guiding gastric cancer clinical therapy by applying the different, sensitive, scientific individual chemotherapy according to the detection results of drug resistance gene, and is helpful to achieve better chemotherapy efficacy for gastric cancer patients.
Key words: Gastric carcinoma; P gp; GST π; Topo Ⅱ; Multiple drug resistance; Chemotherapy
Gastric cancer is a common cancer morbidity and mortality in the country are located in the top of the malignant tumor, treatment when most patients in advanced, lost opportunities for surgical treatment, chemotherapy become the primary means of treatment, but because of resistance generation of drug efficacy is often poor. How to improve the effect of chemotherapy, reducing resistance is the difficulty of clinical treatment. Recent studies indicate that tumor resistance mechanisms mainly P glycoprotein (P gp) overexpression glutathione S transferase π (GST π) activity enhancements and topoisomerase Ⅱ content reduction or change in the nature of (Topo Ⅱ) . Because of P gp, GST π, Topo II resistance mechanism of the same as the corresponding drug resistance are not the same, the clinical treatment should try to use sensitive to chemotherapy drugs. This research attempts by detecting the resistance gene expression, and then select the appropriate sensitive drugs adapted chemotherapy, and to evaluate the effect of treatment of gastric cancer.
1 Materials and Methods
1.1 Clinical data
All 80 patients were in patients with pathologically confirmed gastric endoscopy in our hospital from 2005 to 2007, in which 53 male patients, 27 females; aged 36 to 73 years, average 56 years old; neither line chemotherapy in all cases into groups radiotherapy. Histological type: moderately differentiated adenocarcinoma, 32 cases, other poorly differentiated and undifferentiated adenocarcinoma, signet ring cell carcinoma, 48 cases.
1.2 Inclusion criteria
According to pre-programmed random number will be selected in order of 80 patients were randomly divided into two groups – the adapter group and the non-fit group. Are 40 cases in each group. Age, gender, histological type of the two groups of patients were not significantly different. Inclusion criteria were: (1) patients with gastric cancer pathology diagnosis is based on; (2) is expected to survive for more than three months; (3) Karnofsky functional status (KPS) score ≥ 60; (4) signed consent of chemotherapy.
The 1.3 resistance gene expression detection
Be detected by immunohistochemical methods on paraffin specimens of 80 patients with gastric biopsy. Mouse anti-human P gp, GST π, Topo II monoclonal antibody and SP kit were purchased from Fuzhou Maixin Biotechnology Development Co., Ltd., operating according to instructions. Phosphate buffered saline (PBS, pH = 7.2) instead of the first antibody as negative control, with the known positive colon cancer tissue as a positive control. SP immunohistochemistry method are as follows: (1) conventional slice dewaxing to distilled water; (2) dropping of 3% hydrogen peroxide methanol solution at room temperature for 15min, to deal with the inactivation of endogenous peroxidase, PBS washed 3min × 3 ; (3) is set to 0.01M citrate buffer microwave heating to boiling and after the power outage interval 5min after repeated 1 time, to fix the antigen, after cooling 3min × 3 times washed with PBS; (4) was added dropwise antigen to repair liquid at room temperature 10min, PBS wash 3min × 3 times; (5) each slice dropping 50μl non-immune animal serum at room temperature for 15min, discard the excess liquid, do not wash; (6) dropping the first antibody (anti- P gp, anti-Topo Ⅱ and anti-GST π antibody), wet box 4 ° C overnight (at least 10h) then PBS washed 3min × 3; (7) each slice dropping 50μl biotin-labeled second antibody, 15min, PBS rinse 3min × 3 times; (8) each slice dropping 50μl streptomycin avidin avidin peroxidase solution, 15min, PBS rinse 5min × 4; each section (9) added dropwise a solution of 1 drop of freshly DBA chromogenic agent, at room temperature color under the microscope, controlling the reaction time (observation 3 to 10min), chromogenic satisfied sufficiently washed with tap water; (10) light staining hematoxylin 5 ~~ 10s, immersed in flowing fully washed in tap water; (11) gradient in dry ethanol dehydration, xylene, neutral glue Fengpian, dried at room temperature, optical microscopy positive cells.
1.4 test results to determine
Positive cells were brown finely granular, GST π, Topo Ⅱ, P gp located in the cytoplasm and cell membrane located in the nucleus is located in the nucleus or cytoplasm. Each slice by two doctors, double-blind observation expression in 10 high power field, according to the number of tumor cells positive staining reaction depth determination, no positive expression or the number of positive cells <5% negative (-) The number of positive cells> 5% positive (+).
Adaptation group patients according to the test results of the resistance gene chosen relatively sensitive adaptation chemotherapy for selection of chemotherapeutic agents is divided into the following categories: (1) Plant: paclitaxel, docetaxel, irinotecan; (2) heavy metals class: cisplatin, oxaliplatin platinum; (3) anti-metabolic: 5-Fu, tegafur, capecitabine; (4) synergist: calcium folinate. Specifically chemotherapy drugs are shown in Table 1. Patients with non-adaptive group unified choice FOLFOX4 chemotherapy. 2 patients in addition to chemotherapy symptomatic and supportive treatment measures basically the same observation period auxiliary application of traditional Chinese medicine and immune agents. Each study patients receiving chemotherapy, the time limit of 2 cycle, some indications for surgery patients after 2 cycles transfer surgery, part of the indications for surgery or do not want surgery continue to receive chemotherapy. Table 1 adapter group of 40 patients specific medication and efficacy
1.6 chemotherapy Evaluation
1 week before chemotherapy and two cycles of chemotherapy after 1 week measurement of lesion change. Lesions change refers to the change in the size of tumor lesions by endoscopy, upper gastrointestinal barium meal, CT or MRI to assess. WHO solid tumor efficacy evaluation criteria, divided into complete remission (CR), partial remission (PR), stable (SD) and progress (PD), effective CR + PR.
1.7 statistical methods using χ2 test statistics.
2.1 P gp, GST π, Topo Ⅱ expression rate
P gp, GST π, Topo Ⅱ expression rate were 51.5%, 57.5%, 46.3%; the high differentiated by P gp expression rate (65.6%) was significantly higher than that of other non-moderately differentiated (41.7%) , Topo Ⅱ expression rate (31.3%) is significantly lower than that of other non-high school differentiation (56.3%), (P all <0.05); no significant difference in expression of GST π in high differentiated and non-high school differentiation between see Table 2.3 kinds gene were resistant to 10 cases (12.5%), the two genes were resistant in 35 cases (43.8%), as shown in Table 3. Table and efficient resistance gene expression and tissue differentiation between non-fit group, 35% (14/40), adaptation efficiency of 57.5% (23/40) of the group, which is fully adapted to the patient an effective rate of 64.3%. Two sets of efficiency difference was statistically significant (P <0.05), as shown in Table 4. Table 42 patients chemotherapy efficiency comparison
P gp containing 280 amino acids, with the nature of the channel protein membrane transport proteins. P gp ATP’s participation can be combined with a variety of drugs, and the active energy diffusion into cells lipophilic pump the drug out of the cell, and thereby reduce the concentration of intracellular drug, leading to cell resistance [ 2]. P gp expression positive tumor cells sensitive lipophilic anticancer drug resistance, such as antibiotics, plant alkaloids, alkylating agents, antimetabolites class of chemotherapy drugs . Glutathione S transferase (glutathion s transferase, GST) is a group of multi-functional drug-metabolizing enzymes and intracellular detoxification closely related to the clinical application of many drugs are the GST catalyzed by glutathione ( GSH) combined with detoxification. GST according isoelectric can be divided into three kinds of alkaline, neutral, acidic. Wherein the acidic GST (GST π) is the most common tumor cells and tissues isozyme, is highly expressed in many drug-resistant cells, especially cells of the MDR phenotype. GST primarily by GSH uncoupling the chemotherapy drugs, to increased the toxic drugs outflow reduced cytotoxicity . GST π positive resistant to alkylating agents, anthracyclines, platinum compounds . Topo Ⅱ is a can into another enzyme topological isomers of DNA by a topological isomers, is an important eukaryotic cells indispensable ribozyme. Cutting, rotating and reconnection of DNA function, is also involved in genetic recombination, transcription, sister chromatid separation and DNA repair . Topo Ⅱ chemotherapy drugs target enzymes are mainly two kinds of mechanisms : (1) combine to form a stable fracture complex and Topo Ⅱ DNA to promote DNA fragmentation, and prevents Topo Ⅱ fracture re-connection of the chain, leading to DNA chain scission bug fixes, and kill tumor cells stimulate the biochemical processes of cell death; (2) direct anti tumor effects by inhibiting the activity of Topo Ⅱ. Topo Ⅱ mediated resistance of the foundation that is that the reduction in the content of the Topo Ⅱ and (or) Topo Ⅱ activity reduction. Topo Ⅱ expression the negative natural or semi-natural anti-cancer drugs such as doxorubicin, vinblastine, VP 16, hydroxycamptothecin and other resistance .
Previous studies have shown that P gp, GST π, Topo Ⅱ in gastric cancer have different levels of expression, and biologic behavior of tumors [9 11]. P gp, GST π, Topo Ⅱ in gastric cancer tissues expression rate in the present study were 51.5%, 57.5%, 46.3%; the high differentiated by P gp expression rate (65.6%) was significantly higher than other poorly differentiated, undifferentiated non-high differentiated adenocarcinoma, signet ring cell carcinoma (41.7%), while the expression of Topo Ⅱ (31.3%) was significantly lower than that of other non-high school differentiation (56.3%), suggesting that the two gastric carcinoma histological type, which is more sensitive to anti-cancer drugs with the most poorly differentiated gastric adenocarcinoma phenomenon consistent. GST π expression in high differentiated and non-high school differentiation between different, regardless of histological type of gastric cancer.
The results of this study show that the three kinds of genetic testing results prompted resistance phenotype minority (12.5%), so simply select the appropriate sensitive drug chemotherapy theoretically can increase the effect of cancer chemotherapy. From the results, the adapter patients selected according to the resistance gene expression relatively sensitive to chemotherapy, the overall response rate (57.5%) was significantly higher than the non-fit group FOLFOX4 traditional experience programs (35%), completely adapted have higher efficiency (64.3%).
Based on the above studies, we believe that based on the detection of drug resistance genes results for different patients, the implementation of scientific drugs fit individual chemotherapy for the treatment of gastric cancer has important guiding significance.