Expression Of SSTR2, SSTR5 In Laryngeal Carcinoma And The Inhibited Proliferation Of Laryngeal Carcinoma Cell Hep-2 Induced By Octreotide

Tumor cells have the characteristics of infinite proliferation, blocked differentiation and inhibited apoptosis, which is the significant pathogenesis of malignant tumor. The balance between proliferation and apoptosis are thought to be the important foundation to sustain homeostasis and normal organizational morphous. The breakdown of this balance can ultimately lead to cell cancerization by providing the defected-gene cells with living dominance, promoting gene mutation to assemble and cells malignant transformation. According to the recent researches, somatostatin (SST) and its analog (SSTA) can not only contribute to resisting hyperplasy aiming at many solid tumors and normal tissues, but also inhibiting proliferation and inducing apoptosis of malignant cells, which is mediated by somatostatin receptors (SSTR), and the SSTRs in tumor tissue have much more affinity than those in normal tissue .Laryngeal carcinoma is the most common malignant tumor occurring in larynx. The incidence of laryngeal cancinoma shows an increasing trend because of the environmental pollutions. As same as all other malignancies, various factors contribute to the oncogenesis of laryngeal cancinoma, such as smoking, alcohol, air pollution, viral infection, genetic background, sex hormone and so on. Although the operation technique has been improved greatly, the management of advanced laryngeal cancinoma continues to be a challenging clinical problem. Compared with traditional treatment modalities, chemotherapy given on induction schedules to patients with advanced laryngeal cancinoma allows greater organ preservation without compromise to survival. Our study includes two parts:①expression of SSTR2 , SSTR5 in laryngeal squamous cell carcinoma(LSCC), examination of PI (proliferation index) and AI (apoptosis index) of LSCC, and their relationship with SSTR2, SSTR5.②effects of octreotide on laryngeal carcinoma cells in vitro.1. Expression of SSTR2 and SSTR5 in laryngeal cancinoma and the relationship between their expressionsA total of 46 cases of LSCC, corresponding 30 cases of adjacent tissues of cancer and 20 cases of soft palate tissues as control group were obtained from the department of Otolaryngology, the second clinical hospital of JiLin University, which were examined by reverse transcription-polymerase chain reaction (RT-PCR) and immunochemistry. 38 males, 8 females; age from 36 to 78, 32 cases over 50 ages; glottic type 20, supraglottic type 23, subglottic type 3; 23 cases well-differentiated LSCC, 13 cases moderate and 10 cases poor- differentiated LSCC; stageⅠ:12, stageⅡ:13, stageⅢ:15, stageⅣ:6. The positive expression rates of SSTR2 and SSTR5 were 58.70% and 82.61% in LSCC, 83.33% and 90.00% in adjacent tissues of cancer, 75.00% and 90.00% in control group. There was no significant difference in the positive rate of SSTR5, but the positive rate of SSTR2 in LSCC was significantly lower than that in both adjacent tissue of cancer and control group (P<0.05) . A significant correlation was found between SSTRs expression and tumor stage (P<0.01), tumor differentiation (P < 0. 001). Proliferative index (PI ) and apoptosis index (AI) were examined by immunochemistry and TUNEL. The results showed: PI was in inverse correlation with the expression of SSTRs (P<0.01), and AI was in direct correlation with the expression of SSTRs (P<0.01). So that there were SSTR2 and SSTR5 expressed in many LSCCs, and their expressions were in correlation with proliferation and apoptosis, then SST and SSTA might be used to treat those patients with SSTRs positive expressing.2. Octreotide inhibited the proliferation of laryngeal carcinoma cell Hep-2Effects of octreotide on Hep-2 cells were observed through phase-contrast microscope, cell growth curve, MTT assay and flow cytometry. Under the microscope, apoptotic cells increased as the concentration of octreotide changed, in the environment of 0.1□/ml octreotide, apoptotic cells were most. From 0.01□/ml to 60□/ml, octreotide could inhibit the proliferation of Hep-2 cells, the most effect occurred when octreotide were used at 48 hours. But, compared with the Hep-2 cells of serum-free group , the inhibition of serum group decreased significantly(P<0.05), that might because there were peptidases which could degrade octreotide in fetal calf serum (FCS). In the serum-free group, during 0.01μg/ml～0.1μg/ml, the inhibition increased as octreotide concentration increased, and there were significant differences (P<0.05, vs control). The inhibition of 0.1μg/ml octreotide was most significant, 24.67% (P<0.01, vs control), which showed the effects of octreotide depended on its concentration. Then, with the concentration increased from 0.2□/ml to 60.0□/ml, the effects of octreotide had not increased as well, but their inhibitions had significant difference with the control group, which suggested that the effects of octreotide corresponded to the receptor-saturation theory. According to the results of MTT assay, we used octreotide which concentration were 0.05μg/ml, 0.1μg/ml, 0.5μg/ml, 1.0μg/ml, 10.0μg/ml and 20.0μg/ml to examine the growth curve of Hep-2 cell in serum-free medium. The growth curves of Hep-2 cell depressed significantly (P<0.01, vs control); during 72 hours, the growth curves of Hep-2 cell became flatter with the concentration from 0.05μg/ml to 0.1μg/ml, which was in accord with the results of MTT assay and suggested that octreotide could inhibit the proliferation of Hep-2 cell. The results of FCM: Hep-2 cells showed G0/G1 blcokage , 0.1μg/ml octreotide was the most (73.97±0.48)% (P<0.05, vs control); the cells of G2/M phase decreased (P<0.05 vs control); but the cells of S phase did not have great change(P>0.05 vs control); and apoptosis was also induced, 0.1μg/ml octreotide was the most (2.27±0.15)% (P<0.05, vs control). So the results of FCM suggested octreotide had not affect the DNA synthesis, the inhibition mechanism of Hep-2 cell might be the blockage of cell cycle and apoptosis induced by octreotide, and which effects had time limitation.The default regulation of apoptosis will result in the over proliferation or down apoptosis, and then the malignant potentiality reinforced, that is one of the mechanisms of drugs to treat tumors. G1 phase is the prophase of DNA synthesis,which is the important phase for cell proliferation. According to the clinical studies that SST and SSTA combined with chemotherapeutics will have better effect than their respective use, and the side effects of SSTA are less than chemotherapeutic drugs, so the knowledge of octreotide's effect on cell cycle will be helpful for the formulation of the treatment for laryngeal carcinoma.