Study On Application Of Somatostatin And Its' Receptor In Targeted Therapy Of Human Non-Small Cell Lung Cancer

Death from lung cancer is one of the most common types of cancer-related death. Non-small-cell lung cancer (NSCLC) accounts for approximately 80% of lung tumors. Surgery is not an option for treatment because approximately 65% of these patients have advanced-stage (IIIB/IV) disease at diagnosis. Targeted chemotherapy is a modern approach for the treatment of cancers because it is more efficacious and less toxic than usual systemic chemotherapy. Somatostatin receptor is a peptide receptor, which was researched frequently at preastent. Some investigators have reported that cells of human small cell lung cancer and NSCLC overexpress SSTRs (75-100%). And somatostatin might be a good carrier for the targeted therapy in NSCLC.Paclitaxel possesses excellent antitumor activity in a wide variety of tumor models, especially in NSCLC . In the presence of paclitaxel, depolymerization of the microtubules is inhibited, thereby interfering with the G₂ and M phases of the cell cycle. In spite of its excellent antitumor activity, paclitaxel is not cell specific. Octreotide is an octapeptide analog of endogenous SST and binds to SSTR-2, SSTR-3 and SSTR-5. By virtue of these properties, we thought that octreotide had the potential be a suitable vehicle for delivering paclitaxel to its intracellular target. Therefore, we developed a series of cytotoxic octreotide conjugates that contain paclitaxel and evaluated their cytotoxicity in two NSCLC cell types, namely, Calu-6 cells, which are known to express SSTR and A549 cells, whose expression of SSTR is considered to be low.The expression of SSTR subtypes was influenced by many hormones, such as glucocorticoids, estrogen, thyroxine, insulin, and testosterone. In resent research, the expression of SSTR was thought to be correspondence to the progesterone receptor. Progesterone may up-regulate the expression of SSTR.Progesterone could inhibit the growth of cells by many pathways. Somatostatin was proved to have the anti-tumor effects by direct and indirect ways. The research on regulation of the expression of SSTR subtypes by progesterone and the combined anti-tumor effects of progesterone and somatostain on NSCLC might play an important part in the biotherapy of NSCLC.OBJECTIVE: Inorder to research the effects of somatostatin in the targeting therapy and endocrine therapy NSCLC, we evaluated the effects of the conjugates on the growth of Calu-6 NSCLC cells and A549 NSCLC cells. And A549 cells were chosen to investigate the regulation of SSTR subtype mRNAs expression by progesterone and the combined antiproliferation of progesterone and octreotide.METHODS: The reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect mRNA for human somatostatin receptor subtypes (SSTRs) using specific primers. MTT assay was used to evaluate the cell viability after treating the cells by paclitaxel and the conjugates. Cell cycle perturbations were determined at 24 hours after treatment using FAC Scan flow cytometer. The progesterone receptor was detected by immunohistochemical method, and SSTR subtype mRNAs were determined by RT-PCR in cells pretreated or non-pretreated by progesterone. The viability of A549 cells was evaluated by MTT assay.RESULTS: Both A549 cells and Calu-6 cells expressed the mRNA for SSTR1, SSTR2, SSTR4 and SSTR5. The expression of SSTR2 was strong in A549 cells while and the expression of SSTR5 was strong in Calu-6 cells. No mRNA for SSTR was detected in fibroblast cells.The conjugates retained the biological activity of paclitaxel, and they effectively inhibited the growth of A549 cells and Calu-6 cells in both a concentration-dependent manner and a time-dependent manner. The cytotoxicity of the conjugates was similar to paclitaxel in both A549 cells and Calu-6 cells. Pretreatment with octreotide antagonize the cytotoxicity of the conjugates, but increase the cytotoxicity of paclitaxel in A549 cells.In comparison with free paclitaxel, the conjugates showed much less toxicity in fibroblast cells. The conjugates could increase G2/M fraction in A549 cells just as paclitaxel.Progesterone receptor was detected in A549 cells. The expression of mRNA for SSTR subtypes were upregulated after A549 cells were treated by 0.1nmol/L progesterone for 24 h (p<0.05). Treating A549 cells by 10nmol/L progesterone for 24 h, SSTR3 was detected. Prolonging the treating time to 48h, the expression of mRNA for SSTR3 and SSTR5 were still up-regulated (p=0.012, p=0.02).Both progesterone and octreotide inhibited the growth of A549 cells. The viability of A549 cells treated by progesterone combined with octreotide was lower than that of cells treated by progesterone or octreotide (p<0.05). CONCLUSIONS:1. Both A549 cells and Calu-6 cells expressed the mRNA for SSTR1, SSTR2, SSTR4 and SSTR5.2. The conjugates exerted the same antitumor effect of free paclitaxel on inducing cell death in NSCLC A549 cells and Calu-6 cells. And the conjugates were less toxicity to cells without the expression of SSTR. Our results suggested that paclitaxel conjugated octreotide might prove useful for exploring the efficacy of receptor-directed cytotoxicity to inhibit the proliferation of NSCLC cells, and the conjugates might be used as a targeting agent for NSCLC therapy.3. Progesterone up-regulated the expression of mRNA for SSTR subtypes in A549 cells.4. Progesterone enhanced the ability of octreotide to inhibit the growth of A549 cells. Our results suggested that somatostatin and SSTRs may play an important role in the targeted therapy and endocrine therapy in NSCLC.