Inhibition Suppressed Tumor Growth Research Articles

Abstract A37: Mechanistic study involving antiproliferative effect of Etoricoxib, a highly selective COX-2 inhibitor on human cancer cell lines

Abstract Introduction: Cyclooxygenase (COX), also known as prostaglandin endoperoxidase or prostaglandin G/H synthase, is a rate limiting enzyme involved in the conversion of arachidonic acid to prostaglandins. Two forms of cyclooxygenase, COX-1 and COX-2, have been identified. COX-1 is constitutively expressed in many tissues, while COX-2 is an inducible enzyme involved in inhibition of apoptosis, potentiation of cell growth and angiogenesis and as such is a target for drug development. Recently clinical, cellular and animal experimental studies have indicated its relevance to tumor invasion and metastasis. Etoricoxib is a novel non-steroidal anti-inflammatory drug (NSAIDs) that stops the production of inflammatory prostaglandins, without stopping the production of prostaglandins that protect the stomach and intestine. There is persuasive evidence that COX-2 inhibitors suppress tumor growth due to their ability to inhibit cell proliferation and induce apoptosis. The purpose of this study was to elucidate the mechanism of action by which Etoricoxib induces the arrest of Human cervical tumor cells growth. Methods: Cell viability was measured by MTT assay and trypan blue exclusion method. Apoptosis was examined by DNA fragmentation and Comet Assay and the distribution of cells in the cell cycle was estimated by flow cytometry. Western blotting was used to explore various mechanisms of Etoricoxib induced apoptosis. Results: Etoricoxib in the concentration range of 100–200 µM was 2–3 fold more cytotoxic (MTT assay) in the tumorigenic cell lines HeLa (cervical COX-2 positive), C-33A (cervical COX-2 negative), U-87, MCF-7, Hep3B, MiaPaCa-2 as compared to the non-tumorigenic cell lines (Cos-7, NIH3T3, HEK). These results correlated well with the observations on inhibition of cell growth and were comparable to the results reported for Celecoxib, another COX-2 selective inhibitor. Interestingly, Methotrexate widely used anticancer agent, showed 70–80% cytotoxicity in the non tumorigenic cell lines as compared to only 20% cytotoxicity with etoricoxib, suggesting thereby that it is selectively toxic to tumor cells unlike many other anticancer drugs. Comet assay results in HeLa cells showed a dose and time dependent increase in DNA damage suggestive of drug induced DNA damage at early times, followed by apoptotic DNA fragmentation and necrotic DNA degradation at later times. Western blotting results confirms the downregulation of the NF-κB pathway. In conclusion this study shows that in the human cervical cancer cell lines, HeLa & C33A, Etoricoxib suppresses proliferation, induces apoptosis and causes cell death by DNA damage in both Cox-2 dependent as well as independent pathway. Citation Information: Cancer Res 2009;69(23 Suppl):A37.

Selective inhibition of JNK with a peptide inhibitor attenuates pain hypersensitivity and tumor growth in a mouse skin cancer pain model

Cancer pain significantly affects the quality of cancer patients, and current treatments for this pain are limited. C-Jun N-terminal kinase (JNK) has been implicated in tumor growth and neuropathic pain sensitization. We investigated the role of JNK in cancer pain and tumor growth in a skin cancer pain model. Injection of luciferase-transfected B16-Fluc melanoma cells into a hindpaw of mouse induced robust tumor growth, as indicated by increase in paw volume and fluorescence intensity. Pain hypersensitivity in this model developed rapidly (< 5 days) and reached a peak in 2 weeks, and was characterized by mechanical allodynia and heat hyperalgesia. Tumor growth was associated with JNK activation in tumor mass, dorsal root ganglion (DRG), and spinal cord and a peripheral neuropathy, such as loss of nerve fibers in the hindpaw skin and induction of ATF-3 expression in DRG neurons. Repeated systemic injections of D-JNKI-1 (6 mg/kg, i.p.), a selective and cell-permeable peptide inhibitor of JNK, produced an accumulative inhibition of mechanical allodynia and heat hyperalgesia. A bolus spinal injection of D-JNKI-1 also inhibited mechanical allodynia. Further, JNK inhibition suppressed tumor growth in vivo and melanoma cell proliferation in vitro. In contrast, repeated injections of morphine (5 mg/kg), a commonly used analgesic for terminal cancer, produced analgesic tolerance after 1 day and did not inhibit tumor growth. Our data reveal a marked peripheral neuropathy in this skin cancer model and important roles of the JNK pathway in cancer pain development and tumor growth. JNK inhibitors such as D-JNKI-1 may be used to treat cancer pain.

Blocked pathways: FTIs shut down oncogene signals.

Ras proteins play fundamental roles in cell signal transduction pathways that regulate cell growth, differentiation, proliferation, and survival. ras mutations are among the most frequently encountered genetic abnormalities in human cancers and play a key role in tumorigenesis. The enzymatic attachment of a 15- or 20-carbon moiety to the Ras protein through farnesylation or geranylgeranylation, respectively, is a required step in the proper localization and activation of Ras. Inhibition of the catalytic enzymes, farnesyl transferase and geranylgeranyl transferase, is a novel, mechanism-based, targeted approach to cancer therapy development. Geranylgeranyl transferase inhibitors suppress tumor growth by accumulating cells in the G(1)/S cell cycle phase. One mechanism by which farnesyl transferase inhibitors suppress tumor growth is by inhibiting bipolar spindle formation, thereby blocking progression from prophase to metaphase. Although the exact molecular target responsible for the antitumor activity of farnesyl transferase inhibitors is unclear, at least in some tumor cells, inhibition of phosphoinositide-3-OH kinase/Akt-mediated cell survival pathways may play a critical role. Identifying the farnesylated proteins that are targeted by farnesyl transferase inhibitors and the tumor molecular signatures that dictate which set of patients will respond to farnesyl transferase inhibitors are critical end points for future mechanistic studies.

499 RHO-kinase inhibitor suppresses tumor growth and intrahepatic metastases of hepatocellular carcinoma—In vitro and in vivo study

499 RHO-kinase inhibitor suppresses tumor growth and intrahepatic metastases of hepatocellular carcinoma—In vitro and in vivo study

Inhibitors of protein farnesyltransferase as novel anticancer agents.

This paper describes recent progress in the design, synthesis and biological evaluation of inhibitors for the enzyme protein farnesyltransferase (PFTase). This enzyme plays a critical role in the post-translational modification of a range of different intracellular proteins. In particular, PFTase attaches a farnesyl group to the GTPase Ras whose oncogenically mutated form is found in over 30% of human cancers. As a result PFTase inhibitors have been developed as potential cancer therapeutic drugs either by rational design based on the structure of the CAAX carboxyl terminus of Ras or random screening of chemical libraries or natural products. Some of these inhibitors show remarkable inhibition potency against PFTase at subnanomolar concentrations and >1000-fold selectivity compared to the related enzyme geranylgeranyltransferase-I. Certain of these compounds are highly effective at blocking the growth of human tumors in animal models and are now undergoing clinical trials. However, several issues in the research remain unsolved, including the mechanism by which PFTase inhibitors suppress tumor growth. Although it has been established that PFTase inhibitors block prenylation of Ras in vitro, the results in wholecells and animal studies suggest the possibility that proteins other than Ras are affected.

A selective cyclooxygenase-2 inhibitor suppresses tumor growth in nude mouse xenografted with human head and neck squamous carcinoma cells.

The anti‐tumor effect of a selective cyclooxygenase (COX)‐2 inhibitor, JTE‐522, was examined with the human head and neck squamous cell carcinoma cell line KB. KB cells do not produce prostaglandin (PG)‐E2. In vitro, JTE‐522 induced an increase of G1 phase‐arrested cells, suppression of platelet‐derived growth factor (PDGF) production and inhibition of telomerase activity. No cytotoxic effect was detected. In vivo, the growth of the tumor xenografted into nude mice was significantly suppressed by JTE‐522. Suppression of angiogenesis at the periphery of the tumor, increase of G1‐arrested cells and suppression of telomerase activity were observed, together with an increase of apoptotic cell death in the tumor. Immunological enhancement did not play a role. We concluded that the anti‐tumor effect of JTE‐522 was caused by anti‐angiogenesis action, cell cycle arrest and inhibition of telomerase activity of the tumor cells. These combined effects might induce apoptosis.

Heterogeneous suppression of experimentally induced colon cancer metastasis in rat liver lobes by inhibition of extracellular cathepsin B.

Metastatic rat colon cancer cells but not normal rat hepatocytes showed activity of cathepsin B on their plasma membranes. Activity was visualized in living cells with a new fluorogenic substrate, [Z-Arg]2-cresyl violet, and confocal microscopy. When these cancer cells were injected into the portal vein of rats, the animals developed tumors in the liver in a heterogeneous fashion. Three- to four-fold more tumors were found in the small caudate lobe than in the other three large lobes of the liver. Oral treatment with a selective water-soluble inhibitor of extracellular cathepsin B, Mu-Phe-homoPhe-fluoromethylketone, resulted in 60% reduction of the number of tumors and 80% reduction of the volume of tumors in the three large lobes whereas tumor development was not affected in the small caudate lobe. This study supports the conclusions that (a) extracellular cathepsin B plays a crucial but complex role in liver colonisation by rat colon carcinoma cells in vivo, (b) its selective inhibition suppresses tumor growth heterogeneously in the liver and (c) the caudate lobe of the liver is a relatively large risk factor for tumor development.