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Agonist Contraction Stretch

Gefitinib is a promising agent for the treatment of a wide range of tumor types and shows improved therapeutic efficacy when combined with radiation or chemotherapy in various cell lines and xenografts. A recent highlight is the novel finding of intrinsic importance that a subgroup of patients with NSCLC has specific mutations in the EGFR genes are well correlated with clinical responsiveness to gefitinib. These EGFR mutations lead to increased growth factor signaling and confer susceptibility to gefitinib.On the other hand, overexpression of HER in various cystine cancer cell lines or xenografts increases cytotoxicity andor the antitumor effects of gefitinib. Amplification of the HER gene has been reported in several types of cancer including breast, ovarian, stomach. Of the HER family proteins, gefitinib inhibits EGFRHER phosphorylation with an IC of to nmolL and HER phosphorylation with an IC of. AmolL, suggesting about fold difference in the drug sensitivity for EGFR and HER. HER is deficient in the tyrosine kinase domain and therefore shows no affinity to gefitinib.Because no studies seem to have addressed how HER overexpression modulates sensitivity to gefitinib in human cancer cells, we established isogenic cell lines with or without HER overexpression, in order to determine the mechanism of HER action.After hours, ngmL of EGF or vehicle was added, followed by incubation at jC.The protein content was visualized using horseradish peroxidaseconjugated secondary antibodies followed by enhanced chemiluminescence. One hundred microliters of an exponentially growing cell suspension was seeded into a well plate.The Amiodarone hydrochloride following day, various concentrations of gefitinib were added.After incubation for minutes at room temperature, luminescence was measured using a multilabel counter. Gefitinib was added for hours at jC and cells were then and fixed overnight in ethanol at jC.Fixed cells were resuspended in a propidium iodide solution before incubation at room temperature for hour.Two HER transfectants were isolated after introduction of human HER cDNA into LK cells expressing moderate levels of HER, and very low levels of EGFR and HER.We observed no apparent differences in the growth rates between LK mock and its two HER transfectants under exponential growth conditions in the absence or presence of EGF. The expression levels of HER were similar among the four cell lines.Both LKHER and LKHER cells showed an approximately fold greater sensitivity to the cytotoxic effects of gefitinib than their parental counterparts, when IC values were determined by cell survival assay. Both HER transfectants showed similar sensitivities to gefitinib when assayed by cell survival assay.However, sensitivity was about fold lower in LK HER cells than in PC cells, which harbor inframe deletion mutation of EGFR. Overexpression of HER does not alter growth rate with or without EGF.Immunoreactive proteins were visualized by enhanced chemiluminescence.B, cell growth rates are evaluated in LK and its HER transfectants under normal growth conditions with or without ngmL of EGF.Thus, overexpression of HER seems to alter the sensitivity of LK cells to gefitinib.The presence or absence of EGFR mutations plays a key role in the drug sensitivity of NSCLC cells to gefitinib. Drug sensitivity was determined using a cell viability assay in the absence or presence of different doses of gefitinib.

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