| Summary: | 博士 === 國防醫學院 === 生命科學研究所 === 98 === Renal cell carcinoma (RCC) accounts for 3% of all adult malignancies and clear cell RCC (ccRCC) is major type in renal cancer. The recent estimates of the cancer incidence worldwide about 208,000 new cases of kidney cancer have been diagnosed (1.9% of the world total), and 102,000 persons died because of kidney cancer in 2002. In Taiwan, there are 522 persons died because of kidney cancer in 2007, which is the 14th most common cause of cancer death in men and the 13th in women. Incidence and mortality rates are approximately 1.5 fold as high for men as for women worldwide. clear cell renal cell carcinoma (ccRCC) is the most common kidney cancer (~70%) in adults, which characterized by a high microvascular density produced by tumor-induced neoangiogenesis. Studies have revealed that the major cause of ccRCC is due to loss of the von Hippel-Lindau (VHL) gene, which causes an upregulation of hypoxia-inducible factor-alpha (HIF-alpha) activity, and subsequencely leads to overexpression of VEGF-A. However the clinical targeting drugs which target to VEGFR or VEGF have their limitation for antitumor effect.
For further understanding of the issue of drug induced resistant mechanism against anti-angiogenic therapy, we established an in vivo animal model to further approach the issues which come from clinical therapy.
This model was developed using human RCC cell lines, grown as xenotransplants in severe combined immunodeficient mice, exposed daily to sorafenib in vivo, followed by serial reimplantation and re-exposure to sorafenib, until a resistant phenotype was obtained. These cell lines were used for in vitro and in vivo experiments to further determine the resistant mechanism. According to the result of animal experiment, the xenotransplants in severe combined immunodeficient mice show resistance on tumor growth curve under high dosage of sorafenib treatment. IHC staining of tumor sections show lots of macrophages and neutrophils infiltrated in sorafenib treated tumors. Resistant tumor cells show that phosphorylation level of c-Raf has no effect under high dosage of sorafenib treatment by western blotting. And the RNA level of IL-10 shows clear increase in fourth resistant generation of 786-0 tumor cells.
According to those results, we made a hypothesis that the resistance of tumor comes from the unchanged phosphorylation of c-Raf under high dosage of sorafenib treatment, further activates down-stream signal transduction. It causes cytokine secretion which can promote tumor growth and change tumor microenvironments. It also promotes lots of granulocytes and macrophages infiltrated in tumor site to cause chronic inflammation which can help tumor growth. Therefore tumors generate the resistance to high dosage sorafenib.
Recent clinical study shows that small molecular anti-angiogenic drugs, such as sorafenib, sunitinib and bevacizumab, do not have beneficial effects on single usage. Tumor re-growth rapidly continues after drug treatment stops, even causing metastasis.
Based on our previous study, we found that inflammatory responses in the microenvironment of tumors contributed to tumor drug resistance. In addition, it is observed that structurally abnormal blood vessels in tumors promoted tumor necrosis and increased interstitial fluid pressure, which further impedes the efficacy of drug delivery. Therefore, we further combined another targeting drug which can be activated by â-glucuronidase released from necrotic tumor and inflammatory cells, and thereby killing tumor cells specifically. We further combined anti-angiogenetic therapy to improve the efficacy of drug delivery. By aiming to deliver more targeting drugs that are activated specially in tumor microenvironment, we counteract on the unwanted drug resistance in tumor microenvironment for an enhanced anti-tumor activity.
A potential targeting drug for the present research is 9-aminocamptothecin glucuronide (9 ACG) which is a non-toxic and cell-impermeable glucuronide prodrug. It is suitable for use in combination with anti-angiogenesis treatment in cancer therapy. Based on our previous study, it has been shown that anti-angiogenesis treatment can further improve the delivery and activation of glucuronide prodrugs.
Antiangiogenetic antibody DC101 combined with 9ACG shows synergistic long-term tumor suppression (>125 days) on most mice. Immunohistochemistry staining shows tumors from combined treatment (125 days) has large amounts of granulocytes and macrophages infiltration, increased fibrosis and serious neurosis compared to size matched tumors from PBS treatment. It also provides evidence that the major source of β-glucuronidase which can activate 9ACG comes from dead tumor and immune cells. IHC results also show that those cells contain high levels of β-glucuronidase. This complete result proposes that innate immune cells will accumulate in tumor sites. And these cells which have high levels of β-glucuronidase might promote the selected activation of 9ACG to 9AC in tumor site after DC101 antibody treatment. These data show that innate immune factor plays an important role in long term tumor suppression. We also show that vessel endothelial cells became apoptosis after combined DC101 and 9ACG. This combination also causes the blood vessels to decrease. The sections used come from clinical colon patients which show the similar phenomena of large amounts of granulocyte and microphage infiltration. In conclusion, our data shows synergistic anti-tumor activity under a combination of DC101and 9ACG, and provides an evidence for rewarding value for follow-up effort.
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