Blockade of Signal Transduction through MAP Kinase Pathway Presents a Novel Potential Anti-cancer Agents

15 August, 1999

Blockade of Signal Transduction through MAP Kinase Pathway Presents a Novel Potential Anti-cancer Agents

The pathways of signals from without to within cells have been discovered in the past several years and linked to regulatory events such as in cell proliferation, differentiation and apoptosis. One of the key pathways is the mitogenic signal transduction through the cascade of mitogen-activated protein (MAP) kinase that also includes other transducing molecules such as MAP kinase kinase (MEK) and Raf-1. The MAP kinase signalling, for instance, enhances cyclin D1 for cell proliferation, but also arrests cell growth by increasing expression of the cyclin kinase inhibitor p21 (Cip-1/MDA6/WAF1). The level and duration of MAP kinase expression appears to control this differential effects. The constitutive activation of MAP kinase is associated with many cancer cell lines (pancreas, colon, lung, ovary, kidney ) and primary tumors from various human organs (kidney, colon, lung ), and correlated with the simultaneous expression of MEK and Raf-1 (Hoshino R et al, 1999: uid=9989833).
Recent evidence may be summarized for the molecular mechanism of this MAP kinase pathway. Initially, Ras oncogene product activates Raf1, which in turn phosphorylates and activates MEK1 on 2 distinct Ser residues; Activated MEK1 phosphorylates p44 ERK1 and p42 ERK2 (ERK= extracellular signal-regulated kinase) on Thr183 and Tyr185; These activated MAP kinases phosphorylate a variety of factors such as transcription factors to result in cell proliferation and/or differentiation. ERKs are the only substrates so far for MEK1. This tight selectivity, coupled with the pivotal role of the MAP kinase pathway and enhanced expression of its essential components in tumor cells, suggest a notion that the inhibition of the pathway represents an important route to both radio- and chemo-sensitization of tumor cells and a likely target for pharmacological intervention in proliferative diseases.

With this target in mind, Sebolt-Leopold JS et al (1999: uid=10395327) have developed an in vitro cascade assay system for identifying small-molecule inhibitors of the MAP kinase (MAPK) pathway. Glutathione-S-transferase (GST)-MEK1 and GST-MAPK fusion proteins were prepared from bacterial cells, and used for the sequential phosphorylation of MEK1 to MAPK to MBP (myelin basic protein) in the assay system. The screening led to the discovery of PD 184352 [2-(2-chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzamide] that directly inhibits MEK1 with a 50% inhibitory concentration of 17 nM. This compound did not have substantial inhibitory activity against not only MAPK but also many other kinases, even at a higher concentration (10 uM). In the cell culture model of colon 26 carcinoma where MAPK is highly phosphorylated, PD 184352 reduced the phosphorylation level by 75% at 1 uM as assayed by phosphorylated MAPK-specific antibody. Similar results were also obtained with other tumor cell lines of human origin (pancreatic, cervical, breast, ovarian). Morphologically, colon 26 carcinoma cells no longer appeared rounded but flattened , characteristic of nontransformed epithelial cells, at 0.15 uM of PD 184352. After removal of the compound, the cells resumed the rounded transformed appearance. In vivo, mice bearing subcutaneous implants of colon 26 carcinoma cells were administered ip or orally with PD 184352 at 150 mg/kg. After 1 and 6 hrs of treatment, tumors were excised and phosphorylated MAPK were assayed. The compound completely suppressed the MAPK phosphorylation during the treatment by either route of administration. Preliminary data also indicate that PD 184352 inhibits the dispersion of epithelial cells (HT-29 colon cancer cells) induced by hepatocyte growth factor/scatter factor, suggesting its use against tumor invasiveness and metastasis. Thus the MEK inhibitor described here represents a promising, nontoxic and oral approach to the clinical management of colon cancer.

Many other inhibitors of various signal transduction targets have been developed, and some of them are entering the clinical trials. Cancer chemotherapy more or less depends on the differential growth of the tumor from that of the normal cell, the former being assumed to grow faster. The differential growth might be a useful leverage for some cancer but this assumption may not always be so valid, since some tumors grow slow. Past results indicate clearly that the cancer chemotherapy fails to prolong survival time of the patient, even when the tumor may be reduced in size. The blockers of phosphorylation are new types of anticancer agents, and being nontoxic, cytostatic, and oral in some of them may open a new avenue for the development of antitumor drugs with a novel mechanism of action and may end up being able to extend the survival of the patient with enhanced quality of life.
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