Establishment Of An Etoposide-resistant Human Small Cell Lung Cancer Cell Line H446/VP And Study On Its Drug Resistant Mechanism And Reversal

Objective: Small cell lung cancer (SCLC) is a special histologic type of bronchogenic carcinoma. It's malignancy is highest and prognosis is extremely poor in all types of human lung cancers. Though it may be effectively treated by chemotherapeutics initially in the clinic, but can soon develop the multidrug resistance (MDR). MDR is one of the most important reasons that result in the failure of SCLC chemotherapy. MDR is a special broad spectrum drug resistant phenomenon. Once SCLC develops resistance to a certain anticancer drug, it will resist to many other drugs that are structurally and chemically unrelated. Etoposide (VP-16) is one of the derivatives of podophyllotoxin and belong to DNA topoisomerase II ( Topo II) inhibitor. The inhibition of Topo II is known to be a major mechanism for the action of VP-16 by stabilizing the cleavable complexes and inhibit the rejoin of DNA and result in the damage of DNA that trigger cell to death. As a single agent, VP-16 is probably one of the most active drugs for treating SCLC by exceeding 40% response rates in untreated patients. But it also noted that SCLC therapy with VP-16 is often influenced by MDR.It will be useful for effective treatment of the SCLC and other malignancies to establish an etoposide-resistant human small cell lung cancer cell line and understand its biological characterization. Most of multidrug resistant cell lines were induced by successive exposure to increasing amount of anti-tumor drug in previous studies, which is much difference from the way actually practice in clinical chemotherapy that usually use high dose drugs interval. In the present study, SCLC H446 cells were exposed by high dose of etoposide interval. The new MDR cell line was established and named as H446/VP after 7 months' induction. The morphological feature and growth characters of the cell were observed. Flow cytometry was used to study the cell cycle distribution of H446/VP and its parental cell line. MTT assay was used to study the cross-resistant profile of H446/VP cells. The cell line H446 was established in 1982 and has clear genetic background, stable biological character. We did not find the report of the establishment of etoposide-resistant cell line of H446 after searching relevant literatures widely. So the eastablishment of H446/VP is considered to be valubale for understanding the molecular mechanism of resistance to VP-16 and screening new reversal agents for effective treatment of SCLC.The mechanism of MDR is very complex and many factors may take part in it, the main mechanisms involved in MDR are as follow: 1. Membrane protein that has drug efflux pump, such as: P-glycoprotein (P-gp), lung resistance protein (LRP). 2. The abnormality of enzyme system, such as: Topo II and glytathione S-transferase (GST). 3. Inhibition effect on apoptosis, such as: the excessive expression of bcl-2 or c-myc and so on. VP-16 is one of the Topo II poison inhibitors. The MDR mechanism to VP-16 is more complicated. The quantity and quality change of Topo II, including the Topo II isoenzyme, Topo IIαand Topo IIβ. On the basis of establishment of MDR cell line H446/VP, we detected the expression of P-gp, LRP, Topo IIαandβby immunocytochemical method, in situ hybridization (ISH) and Western blotting for trying to reveal it's MDR mechanism.With the understanding of MDR mechanism, more and more eversal agents were studied. Till now, we cannot find an ideal drug to overcome MDR in clinical practice because most of the relevant drugs have high side effect or expensive cost. The traditional Chinese medicine has wide resources and now more than ten thousand drugs were used, so there is great superiority to find MDR reversal agent in it. Tetramethylpyrazinein (TMP) is one of the effect components of Chinese medicine Chuanxiong that has the activity of calcium passage block. Resently the reversal effect of TMP to leukemia cells was observed. The mechanism of it's reversal effect may be resulted from the surpress of P-gp, but there are many mechanism uncleared. W...