Synergistic Blockade of EGFR and HER2 by New-Generation EGFR Tyrosine Kinase Inhibitor Enhances Radiation Effect in Bladder Cancer Cells.

Blockade of EGFR has been proved useful in enhancing the effect of radiotherapy, but the advantages of new-generation EGFR tyrosine kinase inhibitors (TKI) in radiosensitization are not well known. We used two human bladder cancer cells with wild-type EGFR to study the synergism between irradiation and afatinib (an EGFR/HER2 dual kinase inhibitor) or erlotinib (an EGFR kinase inhibitor). Here, we showed that afatinib has better radiosensitizing effect than erlotinib in increasing cancer cell killing, the percentage of apoptotic cells, and DNA damage. Afatinib is also superior to erlotinib in combining radiation to decrease tumor size, inhibit glucose metabolism, and enhance apoptotic proteins in vivo. Finally, erlotinib suppressed cell growth and induced more DNA damage in bladder cancer cells transfected with HER2 shRNA, but not in control vector-treated cells. In conclusion, concomitant blockade of radiation-activated EGFR and HER2 signaling by a new-generation EGFR TKI better inhibits the growth of bladder cancer cells both in vitro and in vivo. The absence of radiosensitization by EGFR inhibition alone and the greater radiosensitizing effect of EGFR inhibitor in HER2 knocked down cells suggest the synergism between HER2 and EGFR in determining radiosensitivity. The regained radiosensitizing activity of erlotinib implies that with proper HER2 inhibition, EGFR tyrosine kinase is still a potential target to enhance radiotherapy effect in these seemingly unresponsive bladder cancer cells.

Targeting epidermal growth factor receptor/human epidermal growth factor receptor 2 signalling pathway by a dual receptor tyrosine kinase inhibitor afatinib for radiosensitisation in murine bladder carcinoma.

Given the promising control of bladder cancer achieved by combined chemotherapy/radiotherapy with selective transurethral resection, obstacles remain to the treatment of unresectable bladder cancer. The aim of this study was to determine whether targeting epidermal growth factor receptor (EGFR)/human epidermal growth factor receptor 2 (HER2) can radiosensitise a murine bladder tumour (MBT-2) cell line. Cell survival, expression of signal proteins and cell cycle changes in MBT-2 cells treated in vitro and in vivo with afatinib, an irreversible EGFR/HER2 inhibitor, plus radiotherapy were investigated by colony formation assay, Western blot assay and flow cytometry, respectively. Ectopic xenografts were established by subcutaneous injection of MBT-2 cells in C3H/HeN mice. Mice were randomised into 4 groups to receive afatinib (10mg/kg/day on day 1-7) and/or radiotherapy (15Gy on day 4). Positron emission tomography (PET) on day 8 was used to evaluate the early treatment response. Afatinib (200-1000nM) increased cell killing by radiation (0-10Gy). Pre-treatment of irradiated cells with afatinib inhibited radiation-activated HER2 and EGFR phosphorylation. As compared to either treatment alone, the combination increased the level of the cleavage form of poly (ADP-ribose) polymerase, the expression of phospho-γH2AX and the percentage of cells in subG1 phase (indicating enhanced induction of apoptosis), and decreased tumour metabolism and inhibited tumour growth by 64%. Afatinib has therapeutic value as a radiosensitiser of murine bladder cancer cells. The synergism between afatinib and radiation likely enhances DNA damage, leading to increased cell apoptosis.

Putative tumor metastasis-associated genes in human gastric cancer.

Gastric cancer is one of the leading causes of cancer mortality and its malignancy, resulting from disseminated cancer cells of diffuse type, is clinically manifested as metastases to the liver and peritoneum. The aim of the present study was to identify putative tumor metastasis-associated genes in human gastric cancer cells of diffuse type. An MKN45 cell line constitutively expressing green fluorescent protein (MKN45-GFP) was established and selected using the Transwell® system for invasive sublines MKN45-GFP-4, MKN45-GFP-10 and MKN45-GFP-12. MKN45-GFP-10 and MKN45-GFP-12 are highly invasive compared to the others. The mRNA levels were measured with cDNA microarrays and correlated with their invasion abilities in these sublines. Many of the genes identified with a positive or negative correlation are associated with angiogenesis, cell cycle, cytoskeleton and cell motility, protease and cell adhesion, as well as cellular signal transduction. In particular, novel genes without known functions were also noted. RT-PCR and western blot analyses were applied to verify the expression of selective genes. Following orthotopical intraperitoneal implantation, MKN45-GFP-12 demonstrated significantly higher in vivo tumor malignancies than parental MKN45-GFP in ascites induction and liver -invasion in mice. We have identified putative gastric tumor metastasis-associated, as well as novel genes. These genes and their protein products are to be further explored for their functional roles associated with tumor metastasis. The molecular profiles of these identified genes, gene transcripts and proteins in the patient specimens are likely to be useful biomarkers for diagnostic, therapeutic and/or prognostics. Most importantly, they may be used as molecular targets for the discovery of antitumor drugs against human gastric cancer metastasis.

Intravenous paclitaxel against metastasis of human gastric tumors of diffuse type.

PURPOSE: Gastric cancer is one of the leading cancerous diseases worldwide. It is diagnosed often at the advanced stage for which chemotherapy is the main treatment option. The prognosis remains poor for metastatic, especially the diffuse type, gastric cancers. We investigated the efficacy of intravenously administered paclitaxel treating metastases of locally disseminated gastric tumors of diffuse type. METHODS: Transfection of green fluorescent proteins (GFP)-expressing plasmid into human gastric cancer MKN45 cells of diffuse type was performed, and MKN45-GFP cells constitutively expressing GFP were isolated. The MKN45-GFP cells were orthotopically inoculated into the mouse peritoneal cavity, and tumor growth and organ metastases were monitored. Liver metastases were harvested, re-inoculated, monitored for liver metastases again, and harvested for further inoculation. This in vivo selection procedure was repeated to isolate a subline with high metastatic abilities demonstrated by in vitro invasion abilities using Transwell((R)) system. By visualizing the GFP-expressing tumors, the effects of intravenously administered paclitaxel against the growing peritoneally disseminated and metastasized tumors in nude mice without laparotomy were measured. RESULTS: An in vivo selected gastric cancer cell line MKN45-GFP-ip4 with high metastatic ability was established. Its invasion ability was inhibited by paclitaxel treatments in vitro. The growths of metastatic and intraperitoneally disseminated MKN45-GFP-ip4 tumors were significantly suppressed by intravenous paclitaxel treatments in nude mice. CONCLUSIONS: We found that intravenous paclitaxel is active against the metastases of human gastric cancer of peritoneal diffuse type, which warrants further investigations on optimizing the perioperative regimens with intravenous paclitaxel therapy for gastric cancer in patients.