Rajesh L. Thangapazham

Multiple molecular targets in cancer chemoprevention by curcumin

Carcinogenesis encompasses 3 closely associated stages: initiation, progression, and promotion. Phytochemicals are nonnutritive components of plants that are currently being studied in chemoprevention of various diseases for their pleiotropic effects and nontoxicity. Cancer chemoprevention involves the use of either natural or synthetic chemicals to prevent the initiation, promotion, or progression of cancer. Curcumin is the active constituent of turmeric, which is widely used as a spice in Indian cooking. It has been shown to posses anti-inflammatory, antioxidant, and antitumor properties. Curcumin has also been shown to be beneficial in all 3 stages of carcinogenesis. Much of its beneficial effect is found to be due to its inhibition of the transcription factor nuclear factor kappa B (NF-kappaB) and subsequent inhibition of proinflammatory pathways. This review summarizes the inhibition of NF-kappaB by curcumin and describes the recently identified molecular targets of curcumin. It is hoped that continued research will lead to development of curcumin as an anticancer agent.

BENEFICIAL ROLE OF CURCUMIN IN SKIN DISEASES

In recent years, considerable interest has been focused on curcumin a compound, isolated from turmeric. Curcumin is used as a coloring, flavoring agent and has been traditionally used in medicine and cuisine in India. The varied biological properties of curcumin and lack of toxicity even when administered at higher doses makes it attractive to explore its use in various disorders like tumors of skin, colon, duodenum, pancreas, breast and other skin diseases. This chapter reviews the data on the use of curcumin for the chemoprevention and treatment of various skin diseases like scleroderma, psoriasis and skin cancer. Curcumin protects skin by quenching free radicals and reducing inflammation through nuclear factor-KB inhibition. Curcumin treatment also reduced wound-healing time, improved collagen deposition and increased fibroblast and vascular density in wounds thereby enhancing both normal and impaired wound-healing. Curcumin has also been shown to have beneficial effect as a proangiogenic agent in wound-healing by inducing transforming growth factor-beta, which induces both angiogenesis and accumulation of extracellular matrix, which continues through the remodeling phase of wound repair. These studies suggest the beneficial effects of curcumin and the potential of this compound to be developed as a potent nontoxic agent for treating skin diseases.

Green tea polyphenol and epigallocatechin gallate induce apoptosis and inhibit invasion in human breast cancer cells

Currently, there is no effective therapy for estrogen independent breast cancer. MDA-MB-231 is an estrogen receptor negative highly invasive human breast cancer cell line and has been used as a relevant model system to evaluate drugs with chemopreventive potential against highly invasive breast cancer phenotypes. Epidemiological studies though inconclusive have shown that consumption of Green Tea Polyphenols (GTP) reduces the incidence and progression of breast cancer. Green tea is an important source of antioxidants that may be useful for chemoprevention of cancer. Recently published preclinical study from our lab suggested that GTP and EGCG treatment inhibit proliferation and induce apoptosis of MDA-MB-231. In this study, we have evaluated apoptotic and anti-invasive activity of green tea polyphenols(GTP) and its principal constituent Epigallocatechin gallate(EGCG) in MDA-MB-231 human breast cancer cell line. In in-vitro human breast cancer model, EGCG and GTP induced apoptosis and significantly decreased invasion of breast cancer cells. Western blotting of MDA-MB-231 cell lysates from EGCG and GTP treated and untreated control revealed an increase in bax, reduction in bcl2 and PARP cleavage. Quantitative fluorescence labeling resulted in a 24-28% reduction in invasion through matrigel by EGCG and 15-23% reduction by GTP in a dose dependent manner. Focussed microarray analysis and reverse transcriptase polymerase chain reaction and zymogram analysis revealed inhibition of MMP-9 expression by polyphenol treatment. Furthermore, AKT was found to be inhibited both at the RNA and protein level by polyphenol treatment. Moreover EGCG and GTP decreased AKT phosphorylation as found out by western blotting for Phospho-AKT(Ser-473). Beta-catenin level was found to be decreased both in cytoplasm and nucleus. For the first time we report the connection of betacatenin and AKT modulation by GTP and EGCG as a possible mechanism for the induction of apoptosis in human breast cancer cells and also inhibition in their invasive capacity.

Skin regenerative potentials of curcumin

Curcumin, an active constituent of the spice turmeric, is well known for its chemopreventive properties and is found to be beneficial in treating various disorders including skin diseases. Curcumin protects skin by quenching free radicals and reducing inflammation through the inhibition of nuclear factor‐kappa B. Curcumin also affects other signaling pathways including transforming growth factor‐β and mitogen‐activated protein kinase pathway. Curcumin also modulates the phase II detoxification enzymes which are crucial in detoxification reactions and for protection against oxidative stress. In the present review, the biological mechanisms of the chemopreventive potential of curcumin in various skin diseases like psoriasis, vitiligo, and melanoma is discussed. The application of curcumin in skin regeneration and wound healing is also elucidated. We also explored the recent innovations and advances involved in the development of transdermal delivery systems to enhance the bioavailability of curcumin, particularly in the skin. Recent clinical trials pertaining to the use of curumin in skin diseases establishes its benefits and also the need for additional clinical trials in other diseases are discussed.

Conditional reprogramming and long-term expansion of normal and tumor cells from human biospecimens

Historically, it has been difficult to propagate cells in vitro that are derived directly from human tumors or healthy tissue. However, in vitro preclinical models are essential tools for both the study of basic cancer biology and the promotion of translational research, including drug discovery and drug target identification. This protocol describes conditional reprogramming (CR), which involves coculture of irradiated mouse fibroblast feeder cells with normal and tumor human epithelial cells in the presence of a Rho kinase inhibitor (Y-27632). CR cells can be used for various applications, including regenerative medicine, drug sensitivity testing, gene expression profiling and xenograft studies. The method requires a pathologist to differentiate healthy tissue from tumor tissue, and basic tissue culture skills. The protocol can be used with cells derived from both fresh and cryopreserved tissue samples. As approximately 1 million cells can be generated in 7 d, the technique is directly applicable to diagnostic and predictive medicine. Moreover, the epithelial cells can be propagated indefinitely in vitro, yet retain the capacity to become fully differentiated when placed into conditions that mimic their natural environment.

Human TSC2-null fibroblast-like cells induce hair follicle neogenesis and hamartoma morphogenesis

Hamartomas are composed of cells native to an organ but abnormal in number, arrangement or maturity. In the tuberous sclerosis complex (TSC), hamartomas develop in multiple organs because of mutations in TSC1 or TSC2. Here we show that TSC2-null fibroblast-like cells grown from human TSC skin hamartomas induced normal human keratinocytes to form hair follicles and stimulated hamartomatous changes. Follicles were complete with sebaceous glands, hair shafts and inner and outer root sheaths. TSC2-null cells surrounding the hair bulb expressed markers of the dermal sheath and dermal papilla. Tumour xenografts recapitulated characteristics of TSC skin hamartomas with increased mammalian target of the rapamycin complex 1 (mTORC1) activity, angiogenesis, mononuclear phagocytes and epidermal proliferation. Treatment with an mTORC1 inhibitor normalized these parameters and reduced the number of tumour cells. These studies indicate that TSC2-null cells are the inciting cells for TSC skin hamartomas, and suggest that studies on hamartomas will provide insights into tissue morphogenesis and regeneration.

Dissociated Human Dermal Papilla Cells Induce Hair Follicle Neogenesis in Grafted Dermal–Epidermal Composites

Tissue engineered skin substitutes are used in the clinic to treat chronic wounds and burns, and in the laboratory to advance our understanding of wound healing, skin biology, and skin disease. One type of skin substitute, dermal-epidermal composites (DEC), also known as skin equivalents or bilayered living skin constructs, are comprised of dermal fibroblasts embedded in a matrix such as collagen and overlaid with keratinocytes (Veves et al, 2001). DECs promote wound healing (Falanga and Sabolinski, 1999) and have been used to model skin development and diseases (Carretero et al., 2011, Kamsteeg et al., 2011), but their use has been limited by the inability of the skin constructs to regenerate hair follicles (HFs). During embryogenesis, mesenchymal cells signal the overlying epithelium to induce HF formation, and in adults a specialized group of mesenchymal cells, the dermal papilla (DP) cells, have been shown to retain the capacity to induce HF regeneration (Hardy 1992, Reddy et al., 2001, Gharzi et al., 2003). DP cells from rodents induce HFs in a variety of assays (reviewed in Ohyama et al., 2010), but it has been difficult to grow human DP cells that maintain inductive capacity in culture (Higgins et al., 2010). Recent technological advances have enabled the use of human cells to form chimeric HFs, for example by combining human keratinocytes and rodent mesenchymal cells in chamber assays (Ehama et al. 2007), human scalp dermal papilla cells and mouse epidermal keratinocytes in flap grafts (Qiao et al., 2009) or injecting human DP cells, grown as spheroids, together with mouse epidermal cells in reconstitution or “patch” assays (Kang et al., 2012). However, to date, complete and entirely human HFs formed from normal cultured cells have not been reported. Recently, the potential for human hair follicle development in grafted DECs was demonstrated using composites containing human neonatal foreskin keratinocytes (NFK) and fibroblast-like cells derived from tuberous sclerosis skin hamartomas (Li et al., 2011). Therefore, we used the conditions developed in these experiments to test for HF formation in DECs using normal human DP cells. Human DP cells isolated from temporal scalp dermis (Promocell, Heidelberg, Germany) from six donors were propagated in vitro according to manufacturers recommendations. Alkaline phosphatase activity, a DP marker which correlates with hair-inducing capacity (Ohyama et al., 2010), was measured in vitro using the BCIP/NBT substrate (Sigma-Aldrich, St. Louis, MO) on passage 5 DP cells. Alkaline phosphatase activity was variable between samples, with cells from 3 of the donors showing alkaline phosphatase activity in more than 50% of the cells (Table 1). DECs were constructed by combining DP cells with rat tail collagen type 1, adding NFKs on top and bringing the constructs to the air-liquid interface for 2 days before grafting onto female nude mice. Eight weeks after grafting, HFs were observed in mice grafted with the 3 human DP cells with higher alkaline phosphatase activity (Table 1, Figure 1a). HFs had a bulb, dermal sheath, hair matrix and cortex (Figure 1b). Epithelial compartments of the HFs were intact with concentric layers of inner and outer root sheaths, sebaceous glands and hair shaft (Figure 1c-e). Fluorescence in situ hybridization showed the hybridization of a human-specific Alu probe (green) to the nuclei of both epithelial and dermal cells within the graft, including dermal sheath and dermal papilla, confirming their human origin (Figure 1f and g). An antibody reactive with human but not mouse COX IV stained follicular epithelium and dermal papilla/dermal sheath of grafts (Figure 1h). Fluorescence in situ hybridization showed the hybridization of a human-specific, pan-centromeric probe (green) to the nuclei of both epithelial and dermal components (Figure 1i), whereas a human-specific Y-chromosome probe (red) hybridized to nuclei in the epidermis and the follicular epithelium (Figure 1j), consistent with the origin of dermal and epidermal cells from female and male donors, respectively. HFs also stained for markers of specific compartments of a fully developed human HF. Cells in the region of the DP and lower DS displayed alkaline phosphatase activity (Figure 1k), normal reactivity with specific antibodies to human nestin (Figure 1l) and versican (Figure 1m). As expected, anagen HFs had more concentrated immunoreactivity to Ki-67 in the region of the hair matrix relative to the overlying epidermis (Figure 1n). The companion layer as identified by keratin 75 staining was present between the inner and outer root sheaths (Figure 1o). The basal layer of the outer root sheath was immunoreactive for keratin 15, a marker of HF stem cells located in the bulge region (Figure 1p). Figure 1 De novo formation of human hair follicles in grafted dermal-epidermal constructs. Representative H&E stained sections of grafts showing (a) pilosebaceous units, (b) DP and matrix, (c, d) hair shaft, inner and outer root sheath, (d) sebaceous gland, ... Table 1 Human dermal papilla cells with higher alkaline phosphatase activity form hair follicles when combined with neonatal foreskin keratinocytes in grafted dermal-epidermal constructs. In summary, we report that cultured specialized human cells such as DP cells can induce complete pilosebaceous units in vivo in the grafted DEC model. Human HF formation may have been enabled by particular features of our experimental methods, such as the use of DP cells from the temporal scalp, use of an occlusive dressing for a long period after grafting, and a long duration for maturation of the grafts. It is not yet known whether these or other factors are critical to enable human HF formation in DECs, but success using the conditions described appears to require a starting population of DP cells in which the majority show alkaline phosphatase activity. This model could be used to evaluate the trichogenicity of various types of dermal cells in combination with different keratinocyte populations, evaluate hair loss therapies and may be adaptable to examine regeneration of other skin appendages and the formation of skin adnexal neoplasms. Next-generation skin substitutes that promote hair follicle neogenesis are expected to promote healing, normal skin function and appearance, and can be used to study of human HF neogenesis and regeneration with cultured adult cells.

Alteration of skin properties with autologous dermal fibroblasts

Dermal fibroblasts are mesenchymal cells found between the skin epidermis and subcutaneous tissue. They are primarily responsible for synthesizing collagen and glycosaminoglycans; components of extracellular matrix supporting the structural integrity of the skin. Dermal fibroblasts play a pivotal role in cutaneous wound healing and skin repair. Preclinical studies suggest wider applications of dermal fibroblasts ranging from skin based indications to non-skin tissue regeneration in tendon repair. One clinical application for autologous dermal fibroblasts has been approved by the Food and Drug Administration (FDA) while others are in preclinical development or various stages of regulatory approval. In this context, we outline the role of fibroblasts in wound healing and discuss recent advances and the current development pipeline for cellular therapies using autologous dermal fibroblasts. The microanatomic and phenotypic differences of fibroblasts occupying particular locations within the skin are reviewed, emphasizing the therapeutic relevance of attributes exhibited by subpopulations of fibroblasts. Special focus is provided to fibroblast characteristics that define regional differences in skin, including the thick and hairless skin of the palms and soles as compared to hair-bearing skin. This regional specificity and functional identity of fibroblasts provides another platform for developing regional skin applications such as the induction of hair follicles in bald scalp or alteration of the phenotype of stump skin in amputees to better support their prosthetic devices.

Androgen responsive and refractory prostate cancer cells exhibit distinct curcumin regulated transcriptome

Curcumin (diferuloylmethane) is the major active component of turmeric and is being actively investigated for its anti-cancer properties. To better understand the biological mechanisms of the chemopreventive potential of curcumin in prostate cancer, we have evaluated curcumin regulated transcriptome in prostate cancer cells. Hierarchical clustering methods and functional classification of the Curcumin-Gene Expression Response (Cu-GER) showed temporal co-regulation of genes involved in oxidative stress response and growth signaling pathways. Interestingly, C4-2B, androgen independent metastatic prostate cancer cells exhibited attenuated Cu-GER response in comparison to parental androgen dependent and less aggressive LNCaP cells. Androgen Receptor (AR) regulated genes which play critical roles in normal growth and differentiation of the prostate gland, as well as in prostate cancer, were also a part of the Cu-GER. Of note curcumin down-regulated transcript encoded by the potentially causal TMPRSS2-ERG gene fusion, a common oncogenic alteration noted in 50-70% of prostate cancer patients. Further more, expression of EGFR and ERBB2 receptor were found to be down-regulated in curcumin treated LNCaP and C4-2B cells. This report for the first time establishes novel features of Cu-GER in prostate cancer cells of varying tumorigenic phenotypes and provides potentially novel read-outs for assessing effectiveness of curcumin in prostate cancer and likely in other cancers. Importantly, new gene-networks identified here further delineate molecular mechanism(s) of action of curcumin in prostate cancer cells.

Homeopathic Medicines Do Not Alter Growth and Gene Expression in Prostate and Breast Cancer Cells In Vitro

Background: Homeopathy is an alternative medical system practiced in all parts of the world. Although several theories are proposed to explain the mechanisms of action, none are scientifically verified. In this study, the authors investigate the effect of selected homeopathic remedies often used to treat prostate and breast cancer. Materials and Methods: The authors investigated the effect of the homeopathic medicines Conium maculatum, Sabal serrulata, Thuja occidentalis, Asterias, Phytolacca, and Carcinosin on prostate and breast cancer cell (DU-145, LNCaP, MAT-LyLu, MDA-MB-231) growth and on gene expression that regulates apoptosis, using MTT and multiprobe ribonuclease protection assay. Results: None of the homeopathic remedies tested in different potencies produced significant inhibitory or growth-promoting activity in either prostate or breast cancer cells. Also, gene expression studies by ribonuclease protection assay produced no significant changes in mRNA levels of bax, bcl-2, bcl-x, caspase-1, caspase-2, caspase-3, Fas, or FasL after treatment with homeopathic medicines. Conclusions: The results demonstrate that the highly diluted homeopathic remedies used by homeopathic practitioners for cancer show no measurable effects on cell growth or gene expression in vitro using currently available methodologies.