Richard M. Niles

Recent Advances in the Use of Vitamin A (Retinoids) in the Prevention and Treatment of Cancer

Vitamin A, its physiologic metabolites, and synthetic derivatives (retinoids) have been shown to have protective effects against the development of certain types of cancer. In addition, pharmacologic amounts of retinoids have been used with some success in the treatment of a few human tumors. The chemoprevention effect of retinoids is most likely exerted at the tumor-promotion phase of carcinogenesis. Retinoids block tumor promotion by inhibiting proliferation, inducing apoptosis, inducing differentiation, or a combination of these actions. Clinically, isotretinoin (13-cis-retinoic acid) significantly decreases the incidence of second primary tumors in patients with head-and-neck cancer and reduces appearance of non-melanoma skin cancer in patients with xeroderma pigmentosum. Retinoic acid has proved to be an effective treatment for promyelocytic leukemia. However, retinoid resistance limits its use as a single agent. Clinical trials are in progress to determine the efficacy of retinoids in treating other types of cancer such as neuroblastoma and breast carcinoma. The development of receptor-selective retinoids and selective inhibitors of retinoid metabolism may lead to further use of retinoids in both chemoprevention and treatment of cancer.

Resveratrol is a potent inducer of apoptosis in human melanoma cells.

Resveratrol is a plant polyphenol found in grapes and red wine. It has been found to have beneficial effects on the cardiovascular system. Resveratrol also inhibits the growth of various tumor cell lines in vitro and inhibits carcinogenesis in vivo. In this study we examined the effect of resveratrol on growth of two human melanoma cell lines. We found that this plant polyphenol inhibited growth and induced apoptosis in both cell lines, with the amelanotic cell line A375 being more sensitive. The potential involvement of different MAP kinases in the action of resveratrol was also examined. Although resveratrol did not alter the phosphorylation of p38 or JNK MAP kinases in either cell line, it induced phosphorylation of ERK1/2 in A375, but not in SK-mel28 cells. These results suggest that in vivo studies of the effect of resveratrol on melanoma are warranted and that this plant polyphenol might have effectiveness as either a therapeutic or chemopreventive agent against melanoma.

Molecular and physiological actions of quercetin: need for clinical trials to assess its benefits in human disease

There is a growing realization that natural products such as phytochemicals can be used in diets or as supplements to prevent or treat human disease. The disciplines of epidemiology, pharmacognosy, and molecular biology have provided evidence that certain dietary constituents decrease blood pressure, influence immune and neuronal function, affect the incidence of cancer, and ameliorate the abnormal properties of cancer cells. Molecular studies have uncovered the interesting feature that most phytochemicals have multiple modes of action. This review focuses on the flavonoid phytochemical quercetin and describes the myriad of conditions in which quercetin affects a number of physiological processes. Despite the compelling information available, including a number of animal studies, translation of these findings into human clinical trials has been slow. The status of current clinical research on quercetin is summarized, and direction for further research is suggested.

Regulation of expression and activity of four PKC isozymes in confluent and mechanically stimulated UMR-108 osteoblastic cells

The transcript (mRNA), protein levels, enzyme activity, and cellular localization of four protein kinase C (PKC) isozymes identified in rat osteogenic sarcoma cells (UMR‐108) were studied at confluent density and during mechanical stress (cyclic stretch). Western blot analysis indicated that growth to confluent density significantly increased the protein levels of cPKC‐α (11.6‐fold), nPKC‐δ (5.3‐fold), and nPKC‐ϵ (22.0‐fold) but not aPKC‐ζ. Northern blot analysis indicated a significant (2.3‐fold) increase in the 10 kb transcript of cPKC‐α, a slight (1.3‐fold) increase in that of nPKC‐ϵ but no detectable change in that of the remaining isozymes. Enzyme activity assays of the individually immunoprecipitated isozymes yielded detectable kinase activity only for PKC‐α, PKC‐δ, and PKC‐ϵ and only in confluent cells, corroborating the selective increase of these isozymes at confluent density. The UMR‐108 cells showed a dramatic orientation response to mechanical stress with cell reshaping and alignment of the cell long axis perpendicular to the axis of force, remodeling of the actin cytoskeleton, and the appearance of multiple peripheral sites which stained for actin, vinculin, and PKC in separate experiments. Longer term mechanical stress beyond 24 h, however, resulted in no significant change in the mRNA level, protein level, or enzyme activity of any of the four PKC isozymes investigated. The results indicate that there are isozyme‐selective increases in the protein levels of PKC isozymes of osteoblastic UMR‐108 cells upon growth to confluence which may be regulated at the transcriptional or the post‐transcriptional level. The results from UMR‐108 cells support the earlier proposal (Carvalho RS, Scott JE, Suga DM, Yen EH. 1994 . J Bone Miner Res 9(7):999–1011) that PKC could be involved in the early phase of mechanotransduction in osteoblasts through the activation of focal adhesion assembly/disassembly and the remodeling of the actin cytoskeleton.

A factor found in the igg fraction of serum of patients with paraneoplastic bilateral diffuse uveal melanocytic proliferation causes proliferation of cultured human melanocytes

Purpose: To determine if there is a factor in the serum of patients with bilateral diffuse uveal melanocytic proliferation (BDUMP) that causes melanocytic proliferation. Methods: Human melanocytes and melanoma cells were grown and exposed to serum or plasma of patients with BDUMP, other neoplastic conditions, or control media. Preliminary studies using serum were conducted in an unmasked fashion. In addition, IgG-depleted and IgG-enriched plasma was also tested in a similar fashion. Experiments using plasma were conducted triple masked. To show that the proliferation was melanocyte selective, human dermal fibroblasts, keratinocytes, and ovarian cancer cells were treated with plasma of the BDUMP cases or controls, and the effect of this exposure on their proliferation was quantified. Results: At 72 hours, the serum of BDUMP patients caused statistically significant increased proliferation of normal human melanocytes. Further studies at 6 days demonstrated similar findings. In addition, melanocytes grown in BDUMP serum exhibited a disorganized morphology with foci of multilayered cells. Cultured melanoma cells also showed statistically significant increase in growth in serum from BDUMP patients compared with controls. Masked plasma studies further confirmed these findings and showed that the IgG fraction appeared to contain the melanocyte growth-stimulating factor. The human fibroblasts, keratinocytes, and ovarian cancer cells did not show an increase in growth with the BDUMP plasma treatment. Conclusion: Patients with BDUMP have a factor in the IgG fraction that selectively causes melanocyte proliferation. How it causes proliferation of human melanocytes and melanoma cells needs to be further elucidated.

Biomarker and animal models for assessment of retinoid efficacy In cancer chemoprevention

AbstractVitamin A is essential for normal growth and development. Epidemiology and laboratory studies suggest that decreased vitamin A levels and defective metabolism/action may contribute to the genesis of certain cancers. Based on this information, natural and synthetic derivatives of vitamin A (retinoids) have been used for chemoprevention of cancer. Retinoids have had some success in the chemoprevention of leukoplakia and in the decreased incidence of second primaries in head and neck cancer. There is little information on biomarkers that can be used to assess the efficacy of the chemopreventive activity of retinoids. The ability of retinoids to induce RARb has been consistently shown to correlate with the response of cells and tissues to retinoic acid, but few other biomarkers have been certified as indicators of retinoid activity. In light of the failure of the ATBC and CARET clinical intervention trials for chemoprevention of lung cancer, greater use of animal models for chemoprevention studies is necessary. The potential combination of phytochemicals that inhibit DNA methyltransferase activity with retinoids holds promise for more effective chemoprevention of retinoid-unresponsive premalignant lesions.

Vitamin A and cancer

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The use of retinoids in the prevention and treatment of skin cancer

There has been a significant increase in the number of cases of skin cancer diagnosed in the US in the past few years. Thus, it seems appropriate to review the available compounds that might be used in the chemoprevention of these lesions. This review focuses on the retinoids and details their results in clinical trials for treatment and prevention of skin cancer. Side effects of the various retinoid derivatives are also discussed. It is concluded that isotretinoin (13-cis-retinoic acid) is the most effective retinoid for the prevention of non-melanoma skin cancers in high-risk patients in clinical trials. Current basic research is focused on developing receptor-selective retinoids which would have a higher therapeutic index for the treatment and chemoprevention of skin cancer.

PPARα/γ Expression and Activity in Mouse and Human Melanocytes and Melanoma Cells

PurposeWe examined the expression of PPARs and the effects of PPARα and PPARγ agonists on growth of mouse and human melanocytes and melanoma cells.MethodsPPARα,β, and PPARγ mRNA qualitative expression in melan-a mouse melanocytes, B16 mouse melanoma, human melanocytes, and A375 and SK-mel28 human melanoma cells was determined by RT-PCR, while quantitative PPARα mRNA levels were determined by QuantiGene assay. PPARα and PPARγ protein was assessed by Western blotting. The effect of natural and synthetic PPAR ligands on cell growth was determined by either hemocytometer counting or crystal violet assay. PPAR transcriptional activity was determined by a PPRE-reporter gene assay, while knockdown of PPARα expression was achieved by transient transfection of siRNA.ResultsBoth mouse and human melanoma cells produced more PPARα and PPARγ protein compared to melanocytes. PPARα mRNA levels were elevated in human melanoma cells, but not in mouse melanoma cells relative to melanocytes. Silencing of PPARα in human melanoma cells did not alter cell proliferation or morphology. PPARγ-selective agonists inhibited the growth of both mouse and human melanoma cells, while PPARα-selective agonists had limited effects.ConclusionIncreased expression of PPARα in melanoma relative to melanocytes may be a common occurrence, however its biologic significance remains to be determined. PPARγ agonists may be useful for arresting the growth of some melanomas.

Retinoic acid-induced AP-1 transcriptional activity regulates B16 mouse melanoma growth inhibition and differentiation

Retinoic acid (RA) inhibits growth and induces differentiation of B16 mouse melanoma cells. These effects are accompanied by a large increase in PKCα mRNA and protein levels and surprisingly an increase in activating protein‐1 (AP‐1) transcriptional activity. To further investigate the RA‐induced AP‐1 activity we established clones of B16 cells stably expressing an AP‐1‐luciferase reporter gene. Treatment of these clones with phorbol dibutyrate increased AP‐1 activity which peaked at 2–4 h and returned to baseline level by 24 h. In contrast, RA treatment resulted in a slow increase in AP‐1 activity that reached a maximum level at 48 h and was maintained for the duration of the treatment. We tested the importance of the RA‐induced AP‐1 activity by establishing clones which stably express a dominant negative fos gene (A‐fos) and have greatly diminished AP‐1 activity. Growth rates of untreated A‐fos expressing cells were similar to wt B16 and clones not expressing A‐fos. However, clones expressing the dominant‐negative fos had a markedly decreased sensitivity to RA‐induced inhibition of anchorage‐dependent and ‐independent growth. Treatment of wt B16 cells for 48 h with RA increased melanin production by two to fourfold, but this effect was completely lost in the A‐fos clones. The ability of RA to induce RARβ and PKCα expression was retained in A‐fos clones, suggesting that A‐fos was not interfering with RAR transcription activation functions. We tested whether the RA‐induced AP‐1 activity might be mediated by the ERK1/2 MAPK pathway. Inhibition of ERK1/2 phosphorylation stimulated AP‐1 activity, which was not additive to that induced by RA. This finding raises the possibility that this MAPK pathway may be a target of retinoid action. Our observations suggest that AP‐1 transcriptional activity induced by RA likely plays an important role in the biological changes mediated by this retinoid in B16 melanoma cells.