Alejandro García-Carrancá

Mexican medicinal plants used for cancer treatment: Pharmacological, phytochemical and ethnobotanical studies

AIM OF THE STUDY This review provides a summary of Mexican medicinal flora in terms of ethnobotanical, pharmacology, and chemistry of natural products related to anticancer activity. MATERIALS AND METHODS Bibliographic investigation was carried out by analyzing recognized books and peer-reviewed papers, consulting worldwide accepted scientific databases from the last five decades. Mexican plants with attributed anti-cancer properties were classified into six groups: (a) plant extracts that have been evaluated for cytotoxic effects, (b) plant extracts that have documented anti-tumoral effects, (c) plants with active compounds tested on cancer cell lines, (d) plants with novel active compounds found only in Mexican species, (e) plants with active compounds that have been assayed on animal models and (f) plants with anti-cancer ethnopharmacological references but without scientific studies. RESULTS Three hundred plant species belonging to 90 botanical families used for cancer treatment have been recorded, of which only 181 have been experimentally analyzed. The remaining 119 plant species are in use in empirical treatment of diseases consistent with cancer symptomatology. Only 88 of the plant extracts experimentally studied in in vitro cellular models have demonstrated active cytotoxic effects in at least one cancer cell line, and 14 out of the 88 have also been tested in vivo with the results that one of them demonstrated anti-neoplasic effects. A total of 187 compounds, belonging to 19 types of plant secondary metabolites, have been isolated from 51 plant extracts with active cytotoxic effects, but only 77 of these compounds (41%) have demonstrated cytoxicity. Seventeen of these active principles have not been reported in other plant species. However, only 5 compounds have been evaluated in vivo, and 3 of them could be considered as active. CONCLUSION Clearly, this review indicates that it is time to increase the number of experimental studies and to begin to conduct clinical trials with those Mexican plants and its active compounds selected by in vitro and in vivo activities. Also, the mechanisms of action by which plant extracts and their active compounds exert anti-cancer effects remain to be studied.

High Association of Human Papillomavirus Infection with Oral Cancer: A Case-Control Study

BACKGROUND The aim of the present study was to determine the association of high-risk human papillomavirus (HR-HPV) in Mexican individuals with oral squamous cell carcinoma (OSCC) and their association with various risk factors. METHODS We designed a matched case-control study. Cases were individuals with newly diagnosed OSCC, age- and sex-matched with controls (1:4). Demographic and clinical data were obtained; also a self-administered questionnaire about sexual behavior was included. DNA from oral brushing was purified to amplify HPV-DNA through MY09/MY11 and GP5+/GP6+ primers and subsequently subjected to sequencing. Conditional regression models were built to calculate odds ratios (ORs) and 95% confidence intervals (CI). RESULTS Sixty two cases and 248 controls (53.2% males), median age 62 years (Q1-Q3=54-72 years) were included. HPV prevalence was 43.5% in cases and 17.3% in controls (HR-HPV: 37.1% cases, 9.7% controls). The most frequent types in cases were HPV-16 and HPV-18 (55.6 and 18.5%). The presence of HR-HPV was associated with OSCC (OR=6.2; 95% CI: 2.98-12.97) controlling for the most common risk factors. An interaction between smoking and drinking was detected, and family history of cancer was also significant (OR: 3.61; 95% CI=1.44-8.99). Early age at first sexual intercourse and large number of lifetime sexual partners showed an association with HR-HPV (p=0.019 and p=0.033, respectively). CONCLUSIONS Oral HR-HPV was strongly associated with OSCC, suggesting that HPV-16 and -18 are risk factors for oral cancer in Mexican patients. A significant association of tobacco and alcohol was confirmed. In addition, family history of cancer was associated with OSCC. The results underline the role of HPV in OSCC and its multifactorial etiology.

Cancer-initiating cells derived from established cervical cell lines exhibit stem-cell markers and increased radioresistance

BackgroundCancer-initiating cells (CICs) are proposed to be responsible for the generation of metastasis and resistance to therapy. Accumulating evidences indicates CICs are found among different human cancers and cell lines derived from them. Few studies address the characteristics of CICs in cervical cancer. We identify biological features of CICs from four of the best-know human cell lines from uterine cervix tumors. (HeLa, SiHa, Ca Ski, C-4 I).MethodsCells were cultured as spheres under stem-cell conditions. Flow cytometry was used to detect expression of CD34, CD49f and CD133 antigens and Hoechst 33342 staining to identify side population (SP). Magnetic and fluorescence-activated cell sorting was applied to enrich and purify populations used to evaluate tumorigenicity in nude mice. cDNA microarray analysis and in vitro radioresistance assay were carried out under standard conditions.ResultsCICs, enriched as spheroids, were capable to generate reproducible tumor phenotypes in nu-nu mice and serial propagation. Injection of 1 × 103 dissociated spheroid cells induced tumors in the majority of animals, whereas injection of 1 × 105 monolayer cells remained nontumorigenic. Sphere-derived CICs expressed CD49f surface marker. Gene profiling analysis of HeLa and SiHa spheroid cells showed up-regulation of CICs markers characteristic of the female reproductive system. Importantly, epithelial to mesenchymal (EMT) transition-associated markers were found highly expressed in spheroid cells. More importantly, gene expression analysis indicated that genes required for radioresistance were also up-regulated, including components of the double-strand break (DSB) DNA repair machinery and the metabolism of reactive oxygen species (ROS). Dose-dependent radiation assay indicated indeed that CICs-enriched populations exhibit an increased resistance to ionizing radiation (IR).ConclusionsWe characterized a self-renewing subpopulation of CICs found among four well known human cancer-derived cell lines (HeLa, SiHa, Ca Ski and C-4 I) and found that they express characteristic markers of stem cell, EMT and radioresistance. The fact that CICs demonstrated a higher degree of resistance to radiation than differentiated cells suggests that specific detection and targeting of CICs could be highly valuable for the therapy of tumors from the uterine cervix.

Induction of p53 protein expression by sodium arsenite

Arsenic is carcinogen for humans and has been shown to act as an enhancer in initiated animal models. In a previous work we found impairment of lymphocyte proliferation in arsenic-exposed individuals and in vitro we obtained dose-related inhibition of mitotic response and lymphocyte proliferation. Intrigued by these effects and based on the role of p53 on cell proliferation, we tested different concentrations of sodium arsenite for their ability to induce the expression of tumor suppressor gene p53 in different cell lines (HeLa, C-33A. Jurkat) and a lymphoblast cell line transformed with Epstein-Barr virus (LCL-EBV). We also evaluated changes in their viability after 24 h arsenic treatment; C-33A cells showed the higher sensitivity to arsenic treatment while HeLa, Jurkat and LCL-EBV cells showed similar cytotoxicity curves. Immunoblots showed an increased expression of p53 gene with 1 microM sodium arsenite in Jurkat cells and 10 microM sodium arsenite in HeLa and LCL-EBV cells. In addition, we transfected Jurkat cells and human lymphocytes with wild-type and mutated p53 genes; lymphocytes and Jurkat cells that received the mutated p53 showed increased sensitivity to arsenic cytotoxicity. Data obtained indicate that arsenic induces p53 expression and that cells with a functional p53 contend better with damage induced by this metalloid.

Transcription-independent triggering of the extrinsic pathway of apoptosis by human papillomavirus 18 E2 protein.

Cervical carcinomas are most frequently associated with human papillomaviruses (HPV), whose E6 and E7 oncogenes products induce cellular immortalization. The papillomavirus E2 protein is a transcription factor, which represses the expression of the viral oncogenes, and activates viral DNA replication during the vegetative viral cycle. This protein is specifically inactivated in HPV18-associated carcinoma cells, suggesting that E2 functions prevent carcinogenic progression. Indeed, ectopic expression of E2 in cervical carcinoma cells strongly inhibits cell proliferation. Here we show that above a threshold level of expression, the E2 protein induces apoptosis, independently of other viral functions. The amino-terminal domain is responsible for this apoptotic activity, but surprisingly with no involvement of its transcriptional functions. The death pathway triggered by E2 relies on activation of the initiator caspase 8, specific of the extrinsic pathway of apoptosis. E2 itself is cleaved by caspases during cell death, providing an example of an apoptotic inducer that is itself a target for caspase processing. The autonomous proapoptotic activity of HPV18 E2 described here may counteract the proliferative functions of viral oncogenes, and renders the inactivation of E2 crucial for carcinogenic progression.

Association between high-risk human papillomavirus DNA load and precursor lesions of cervical cancer in Mexican women

OBJECTIVE Our objective was to determine the association between viral load of high risk human papilloma virus (HPV) using the Hybrid Capture II (HC II) system and cervical intraepithelial neoplasia (CIN) lesion stage. METHODS A total of 182 consecutive women with confirmed diagnoses of CIN 1-3 and 182 healthy women with negative Pap were included. All subjects underwent structured interviews focused on socioeconomic and reproductive factors. HC II testing was used to detect human papilloma virus (HPV) DNA. Viral load was measured by light measurements expressed as relative lights unit (RLU) ratio (specimens/control). Log(10)RLU ratios were categorized for analysis into four groups: negative (</=0); low viral load (0.01-1.0), middle viral load (1.01-2.0), and high viral load (2.0-3.6). Frequencies and association measurement odds ratio (OR) adjusted by unconditional multinomial regression (UMR) were used in analysis. RESULTS A total of 75 of 80 (93.7%) patients with CIN 2-3, 82 of 101 (79.4%) with CIN 1, and 36 of 182 (19.8%) controls were positive for HPV DNA. The higher the viral load of HPV DNA infection observed, the higher the probability of being associated with stage of CIN (P <0.001). Association between low viral load HPV and CIN 1 was 16.8 (7.2-39) compared with the highest association observed with high viral load and CIN 2-3 (OR(a) = 365.8, 94.7-1412). Both control and cases in the oldest women presented the highest viral load. CONCLUSIONS We found high frequencies of HPV DNA in CIN 1 and in CIN 2-3 patients. A clear association between viral load of HPV DNA was determined by HC II assay and CIN stage.

High-risk human papilloma virus infection decreases the frequency of dendritic Langerhans' cells in the human female genital tract

Dendritic cells (DC) are often arranged in planar layers in tissues with high antigenic exposure, such as skin and mucosae. Providing an en face view, this arrangement optimizes in situ analysis regarding morphology (even of individual dendrites), topographic distribution (regular/clustered) and quantification. The few reports on human genital DC usually utilize single markers and conventional sections, restricting immunolabelling only to cell parts sectioned by the cut. To better assess DC in situ, we labelled epithelial sheets, prepared from fresh cervix biopsies, with antibodies to major histocompatibility complex (MHC)‐CII, CD1a and Langerin, revealing (with each of these markers) a dense DC network in a planar‐like, regular distribution. Using the hybrid capture system to detect the high‐risk mucotropic human papilloma virus (HPV) group, 16 positive and five negative women were studied and the results were compared between these groups. DC frequency per area was substantially reduced (to ≈ 50% for the three markers) in samples from all HPV‐infected patients compared with samples from controls. Unlike HPV– samples, Langerin+ DC in HPV+ cervix exhibited a highly accentuated dendritic appearance. We believe this to be the first study using these three DC‐restricted markers (Langerin, CD1a and MHC‐CII) in cervical epithelial sheets from high‐risk HPV+ donors and also the first study to demonstrate the morphological and quantitative changes triggered by high‐risk HPV infection. Cervical DC reduction in early, premalignant high‐risk HPV infection might represent viral subversion strategies interfering with efficient antigen handling by the immune systems peripheral sentinels, the DC, perhaps hampering appropriate recruitment and subsequent development of effector (cytotoxic) T cells.

HPV-related Carcinogenesis: Basic Concepts, Viral Types and Variants

Human papillomavirus (HPV) constitutes a diverse group of small DNA virus, some extensively studied during the last three decades due to their carcinogenic potential. Persistence of viral infections and uncontrolled expression of E6 and E7 viral oncogenes are critical events in transformation. A surprisingly large number of different HPV types have been identified and classified (>100) and it has been anticipated that almost 200 may exist. HPV types are thought to have originated very early during human evolution and are now defined by their L1 genomic sequence, differing by >10% among them. Importantly, viral types are cell-type specific and usually produce different kinds of lesions, benign or malignant. In addition, these types have co-evolved with their hosts and have generated what we call now intratype variants. Variants of HPV types are found associated with the ethnicity of the populations and have been grouped geographically. It is believed that HPV intratype variants may differ in biological behavior. Recognition of the crucial role that some specific HPV types play in cervical cancer development is highly important for their prevention and implementation of public health strategies to control cervical cancer, still the leading cause of death among cancer patients in many developing nations. Here we review basic concepts of HPV-induced carcinogenesis and molecular differences found among HPV types and intratype variants and discuss their clinical and functional implications.

Magnolia dealbata Zucc and its active principles honokiol and magnolol stimulate glucose uptake in murine and human adipocytes using the insulin-signaling pathway.

Some Magnolia (Magnoliaceae) species are used for the empirical treatment of diabetes mellitus, but the antidiabetic properties of Magnolia dealbata have not yet been experimentally validated. Here we report that an ethanolic extract of Magnolia dealbata seeds (MDE) and its active principles honokiol (HK) and magnolol (MG) induced the concentration-dependent 2-NBDG uptake in murine 3T3-F442A and human subcutaneous adipocytes. In insulin-sensitive adipocytes, MDE 50 μg/ml induced the 2-NBDG uptake by 30% respect to insulin, while HK and MG, 30 μM each, did it by 50% (murine) and 40% (human). The simultaneous application of HK and MG stimulated 2-NBDG uptake by 70% in hormone-sensitive cells, on which Magnolia preparations exerted synergic effects with insulin. In insulin-resistant adipocytes, MDE, HK and MG induced 2-NBDG uptake by 57%, 80% and 96% respect to Rosiglitazone (RGZ), whereas HK and MG simultaneously applied stimulated 2-NBDG uptake more efficiently than RGZ (120%) in both murine and human adipocytes. Inhibitors of the insulin-signaling pathway abolished the glucose uptake induced by Magnolia dealbata preparations, suggesting that their antidiabetic effects are mediated by this signaling pathway. In addition, MDE, HK and MG exerted only mild to moderate proadipogenic effects on 3T3-F442A and human preadipocytes, although the combined application of HK and MG markedly increased the lipid accumulation in both cell types. In summary, Magnolia dealbata and its active principles HK and MG stimulate glucose uptake in insulin-sensitive and insulin-resistant murine and human adipocytes using the insulin signaling pathway.

Frequent Alterations of the β-Catenin Protein in Cancer of the Uterine Cervix

Cancer of the uterine cervix is still the leading cause of death among women with cancer in developing countries. Although infections with human papillomavirus are necessary, other molecular alterations that are needed at the cellular level for development of these tumors remain largely unknown. β-Catenin is a key regulator located within the Wnt signaling cascade whose alterations constitute an important event in colon carcinogenesis. In many malignancies increased levels of the β-catenin protein have been found, associated with its nuclear and/or cytoplasmic accumulation. To search for possible alterations of this pathway we examined the expression and localization of the β-catenin protein in tumors from the uterine cervix and cell lines derived from them. β-Catenin was found accumulated in the cytoplasm and/or nuclei of 12 out of 32 samples. In accordance, increased levels of this protein were observed in 9 out of 20 tumors analyzed. Importantly, PCR-SSCP and sequence analysis showed no mutations in exons 3, 4 and 6 of the β-catenin gene. Our findings indicate that alterations of β-catenin are frequent in these tumors and suggest that they may play an important role in the development of cancer of the uterine cervix. They also indicate that higher protein levels and abnormal localization may result from several different mechanisms.