John S. Bertram

The molecular biology of cancer

The process by which normal cells become progressively transformed to malignancy is now known to require the sequential acquisition of mutations which arise as a consequence of damage to the genome. This damage can be the result of endogenous processes such as errors in replication of DNA, the intrinsic chemical instability of certain DNA bases or from attack by free radicals generated during metabolism. DNA damage can also result from interactions with exogenous agents such as ionizing radiation, UV radiation and chemical carcinogens. Cells have evolved means to repair such damage, but for various reasons errors occur and permanent changes in the genome, mutations, are introduced. Some inactivating mutations occur in genes responsible for maintaining genomic integrity facilitating the acquisition of additional mutations. This review seeks first to identify sources of mutational damage so as to identify the basic causes of human cancer. Through an understanding of cause, prevention may be possible. The evolution of the normal cell to a malignant one involves processes by which genes involved in normal homeostatic mechanisms that control proliferation and cell death suffer mutational damage which results in the activation of genes stimulating proliferation or protection against cell death, the oncogenes, and the inactivation of genes which would normally inhibit proliferation, the tumor suppressor genes. Finally, having overcome normal controls on cell birth and cell death, an aspiring cancer cell faces two new challenges: it must overcome replicative senescence and become immortal and it must obtain adequate supplies of nutrients and oxygen to maintain this high rate of proliferation. This review examines the process of the sequential acquisition of mutations from the prospective of Darwinian evolution. Here, the fittest cell is one that survives to form a new population of genetically distinct cells, the tumor. This review does not attempt to be comprehensive but identifies key genes directly involved in carcinogenesis and demonstrates how mutations in these genes allow cells to circumvent cellular controls. This detailed understanding of the process of carcinogenesis at the molecular level has only been possible because of the advent of modern molecular biology. This new discipline, by precisely identifying the molecular basis of the differences between normal and malignant cells, has created novel opportunities and provided the means to specifically target these modified genes. Whenever possible this review highlights these opportunities and the attempts being made to generate novel, molecular based therapies against cancer. Successful use of these new therapies will rely upon a detailed knowledge of the genetic defects in individual tumors. The review concludes with a discussion of how the use of high throughput molecular arrays will allow the molecular pathologist/therapist to identify these defects and direct specific therapies to specific mutations.

Effects of Lycopene Supplementation in Patients with Localized Prostate Cancer

Epidemiological studies have shown an inverse association between dietary intake of lycopene and prostate cancer risk. We conducted a clinical trial to investigate the biological and clinical effects of lycopene supplementation in patients with localized prostate cancer. Twenty-six men with newly diagnosed prostate cancer were randomly assigned to receive a tomato oleoresin extract containing 30 mg of lycopene (n = 15) or no supplementation (n = 11) for 3 weeks before radical prostatectomy. Biomarkers of cell proliferation and apoptosis were assessed by Western blot analysis in benign and cancerous prostate tissues. Oxidative stress was assessed by measuring the Peripheral blood lymphocyte ONA oxidation product 5-hydroxymethyl-deoxyuridine (5-OH-mdU). Usual dietary Intake of nutrients was assessed by a food frequency questionnaire at baseline. Prostatectomy specimens were evaluated for pathologic stage, Gleason score, volume of cancer, and extent of high-grade prostatic intraepithelial neoplasia. Plasma levels of lycopene, insulin-like growth factor-1, insulin-like growth factor binding protein-3, and prostate-specific antigen were measured at baseline and after 3 weeks of supplementation or observation. After intervention, subjects in the intervention group had smaller tumors (80% vs 45%, less than 4 ml), less involvement of surgical margins and/or extra-prostatic tissues with cancer (73% vs 18%, organ-confined disease), and less diffuse involvement of the prostate by high-grade prostatic intraepithelial neoplasia (33% vs 0%, focal involvement) compared with subjects in the control group. Mean plasma prostate-specific antigen levels were lower in the intervention group compared with the control group. This pilot study suggests that lycopene may have beneficial effects in prostate cancer. Larger clinical trials are Warranted to investigate the potential preventive and/or therapeutic role of lycopene in prostate cancer.

Bioactive carotenoids: potent antioxidants and regulators of gene expression.

Abstract Carotenoi ds are plant pigments, some of which act as a vital source of vitamin A to all animals, that appear to have additional benefits to primates. They are potent antioxidants and photoprotectants and can additionally modulate gene activity resulting in protection from experimentally-induced inflammatory damage and neoplastic transformation. Anti-neoplastic properties appear tightly correlated to their ability to induce the gap junctional protein connexin 43 (Cx43). This when upregulated leads to decreased proliferation and decreased indices of neoplasia in animal and human cells. Delivery of natural carotenoids can be compromised by poor bioavailability. To overcome this, a synthetic water-dispersible derivative of astaxanthin has been synthesized and shown to be: highly bioavailable; a potent antioxidant; protective against experimental ischemia-reperfusion injury and capable of inducing Cx43, suggesting antineoplastic potential. The ability to deliver biologically active carotenoids at high concentration and with good reproducibility appears to have been achieved.

Metabolites of dietary carotenoids as potential cancer preventive agents

There is abundant epidemiological evidence that consumption of dietary carotenoids reduces the risk of cancer, but it is unclear which of the more than 24 carotenoids, including 8 metabolites, found in human plasma is active. Here we provide evidence that 3 major dietary carotenoids, beta-carotene, lutein and lycopene, can increase connexin 43 gene expression in 10T112 cells and in human keratinocytes in organotypic culture. This activity is shared with all-trans retinoic acid and is limited to suprabasal cells as is expression of this gene in intact human skin. Furthermore, (3R, 3R )-zeaxanthin and 2,6-cyclolycopene-l,5 diol, metabolic derivatives found in human serum of lutein and lycopene respectively, exhibit greater activity than the parent compounds. We suggest that the conversion of dietary carotenoids to compounds which can increase gap junctional communication may play a role in the protective action of carotenoid- rich foods.

Inducible expression of the gap junction protein connexin43 decreases the neoplastic potential of HT‐1080 human fibrosarcoma cells in vitro and in vivo

Numerous studies have demonstrated a correlation between dysregulation/loss of connexin expression or gap junction intercellular communication (GJIC) function and decreased growth control both in human tumors and tumor cell lines. Likewise, restoration of constitutive connexin expression/function is correlated with increased growth control/decreased tumorigenicity. Here, we show for the first time that inducible restoration of connexin43 (Cx43) expression and GJIC function in a human tumor line of mesenchymal origin (HT‐1080, fibrosarcoma) resulted in a lowered neoplastic potential. Specifically, HT‐1080 cells induced to express Cx43 demonstrated diminished foci formation when in co‐culture with normal fibroblasts, decreased colony formation under anchorage‐independent conditions, and reduced tumor growth when injected into immunodeficient mice. These results, obtained utilizing an inducible system that helps address issues of clonal heterogeneity, strongly implicate Cx43 as a tumor suppressor in human tissue of mesenchymal origin and GJIC as a regulatory mechanism for cellular growth control both in vitro and in vivo. This study also further supports the hypothesis that loss of Cx43/GJIC in human tumors may play an important role in the dysregulation of normal growth control.

14 – Dietary Carotenoids and their Metabolites as Potentially Useful Chemoprotective Agents against Cancer

Publisher Summary nThis chapter emphasizes the importance of other dietary carotenoids in addition to β-carotene, and presents evidence of the nutritional significance of this important class of nutrients in the prevention of cancer as well as macular degeneration, a degenerative eye disease. This was initially based on the knowledge of widespread distribution of carotenoids in fruits and vegetables, as well as the consistent presence of these compounds and their metabolites in human serum, milk, and tissues at relatively high concentrations. Carotenoids are among the most widespread of the naturally occurring groups of pigments and are found in all families of the plant and animal kingdoms. The correlation between dietary carotenoids and carotenoids found routinely in the extracts of human serum/plasma has revealed that only selected groups of carotenoids make their way into the human bloodstream. Some of these carotenoids are absorbed intact and others, such as lutein, zeaxanthin, and lycopene, are converted to several metabolites. Some dietary carotenoids are also present in human tissues, for example, the liver, lung, breast, and cervix. This chapter identifies several new optical isomers of carotenoid metabolities in human plasma, and provides additional evidence of previously proposed metabolic oxidation–reduction reactions of dietary carotenoids in humans. It also presents studies that indicate that carotenoids, in addition to their antioxidant mechanism of action, can exert their biological activity in disease prevention by other mechanisms.

Transcriptional regulation of connexin 43 expression by retinoids and carotenoids: Similarities and differences

Gap junctions, connexons, are formed by assembly of trans‐membrane connexin proteins and have multiple functions including the coordination of cell responses. Most human tumors are deficient in gap junctional communication (GJC) and restoration of GJC by forced expression of connexins reduces indices of neoplasia. Expression of connexin 43 (Cx43), the most widely‐expressed connexin family member, is upregulated by cancer‐preventive retinoids and carotenoids in normal and preneoplastic cells; an action considered of mechanistic significance. However, the molecular mechanism for upregulated expression is poorly understood. The retinoic acid receptor antagonist Ro 41‐5253 was capable of suppressing retinoid‐induction Cx43 luciferase reporter construct in F9 cells, but did not suppress reporter activity induced by the non‐pro‐vitamin A carotenoids astaxanthin or lycopene, indicating that retinoids have separate mechanisms of gene activation than non‐pro‐vitamin A carotenoids. Neither class of compound required protein synthesis for induction of Cx43 mRNA, nor was the 5.0 h half‐life of Cx43 mRNA altered, indicating direct transcriptional activation. The responsive region was found within −158 bp and +209 bp of the transcription start site; this contains a Sp1/Sp3 GC‐box to which Sp1 and Sp3 were bound, as revealed by electrophoretic mobility shift assays (EMSA), but no retinoic acid response element (RARE). Site directed mutagenesis of this GC‐box resulted in increased basal levels of transcription and loss of responsiveness to a synthetic retinoid. In this construct astaxanthin and lycopene produced marginally, but not significantly higher, reporter activity than the control.

Lycopene in the treatment of prostate cancer

Dietary intake of lycopene is associated with reduced risk of prostate cancer (PCa). We conducted a clinical trial in men with prostate cancer to investigate the biological and clinical effects of lycopene supplementation. Twenty-six men with prostate cancer were randomly assigned to receive a lycopene supplement or no supplement for three weeks before radical prostatectomy. Subjects in the intervention group (n = 15) were instructed to take a tomato oleoresin extract soft gel capsule (Lyc-O-Mato®, LycoRed Company, Beer Sheva, Israel) containing 15 mg lycopene, 1.5 mg phytoene, 1.5 mg phytofluene, and 5 mg tocopherol twice daily with meals. Prostatectomy specimens were evaluated for pathologic stage, Gleason score, volume of cancer, and extent of high-grade prostatic intraepithelial neoplasia (HGPIN). Biomarkers of cell proliferation and apoptosis were assessed by Western blot analysis in benign and cancerous tissue samples obtained from the prostatectomy specimens. Oxidative stress was assessed by measuring the peripheral blood lymphocyte DNA oxidation product 5-hydroxymethyl-deoxyuridine (5-OH-mdU). Plasma levels of lycopene, insulin-like growth factor-1 (IGF-1), insulin-like growth factor binding protein-3 (IGFBP-3), and prostate-specific antigen (PSA) were measured at baseline and after three weeks of study period. After the intervention, more men in the intervention group had smaller (<4 cc) tumors, organ-confined disease without involvement of surgical margins or extra-prostatic tissues, and focal involvement of the prostate with HGPIN compared to the control group. Mean plasma PSA levels were lower in the intervention group compared to the control group. This pilot study suggests that a tomato extract containing lycopene and other tomato carotenoids and phytochemicals may have a potential role in the treatment of prostate cancer. Larger clinical trials are necessary to definitively address potential uses of lycopene or tomato extract in the prevention or treatment of prostate cancer.

Reduction in the formation of carcinogen-induced transformed foci by penicillin g sodium in the c3h/10t1/2 cl8 cell line.

The C3H/10T1/2 CL8 cell line is being widely used to study mechanisms of malignant transformation in vitro. As currently employed, the standard assay system uses a combination of penicillin (100 I.U./ml) and streptomycin (50 micrograms/ml) to reduce the occurrence of bacterial contamination. The penicillin component of this mixture has been discovered to cause a reduction in the number of transformed foci which develop after exposure of cells to MCA, DMBA and X-rays. This reduction is dose dependent; 500 I.U./ml virtually eliminates transformation, while 100 I.U./ml causes an approximate 50% decrease in the number of foci. This effect does not appear to be due to overt toxicity and is largely reversible on removal of the antibiotic. Gentamicin (25 micrograms/ml) causes no reduction in the formation of transformed foci when compared to cultures maintained in antibiotic-free medium and offers the advantages of chemical stability, a wider spectrum of antibacterial activity in comparison with penicillin/streptomycin and, in addition, is active against many mycoplasma. It is suggested that future studies with this cell line should ideally be performed without antibiotics or should employ Gentamicin for antibacterial protection.

Cloning and Functional Expression of a Novel Human Connexin-25 Gene

Gap junctions arc intercellular, water-filled channels composed of transmembrane proteins called connexins, six of which are arranged radially and dock with six homologous proteins in an adjacent cell to form an approximate 16 A pore. Through this pore cell-to-cell transfer of small water-soluble molecules up to about 1000 daltons occurs along concentration gradients. Connexins comprise a multigene family that share consensus sequences in the trans-membrane domains and the first and second extracellular loops. Comparison of the protein sequences of known human connexins with the draft nucleotide sequence of the human genome revealed two clones from chromosome 6 which showed strong similarity to highly conserved connexin sequences. Detailed analysis revealed the presence of a 672 nt open reading frame in these clones, encoding a 223 amino acid polypeptide with a predicted molecular weight of about 25 kD. This is smaller than other known human connexins. The ORF of the potential connexin25 was amplified by semi-nested PCR using human genomic DNA as a template. To confirm that this new gene encodes a connexin, Cx25 was transfected into a gap junction deficient subclone of the human HeLa cell line. After selection of transformants, cells were microinjected with the fluorescent dye Lucifer yellow. Transfectants but not controls successfully transferred dye, demonstrating that this new gene encodes a functional connexin.