Curcumin Chemosensitizes 5-Fluorouracil Resistant MMR-Deficient Human Colon Cancer Cells in High Density Cultures

Objective Treatment of colorectal cancer (CRC) remains a clinical challenge, as more than 15% of patients are resistant to 5-Fluorouracil (5-FU)-based chemotherapeutic regimens, and tumor recurrence rates can be as high as 50–60%. Cancer stem cells (CSC) are capable of surviving conventional chemotherapies that permits regeneration of original tumors. Therefore, we investigated the effectiveness of 5-FU and plant polyphenol (curcumin) in context of DNA mismatch repair (MMR) status and CSC activity in 3D cultures of CRC cells. Methods High density 3D cultures of CRC cell lines HCT116, HCT116+ch3 (complemented with chromosome 3) and their corresponding isogenic 5-FU-chemo-resistant derivative clones (HCT116R, HCT116+ch3R) were treated with 5-FU either without or with curcumin in time- and dose-dependent assays. Results Pre-treatment with curcumin significantly enhanced the effect of 5-FU on HCT116R and HCR116+ch3R cells, in contrast to 5-FU alone as evidenced by increased disintegration of colonospheres, enhanced apoptosis and by inhibiting their growth. Curcumin and/or 5-FU strongly affected MMR-deficient CRC cells in high density cultures, however MMR-proficient CRC cells were more sensitive. These effects of curcumin in enhancing chemosensitivity to 5-FU were further supported by its ability to effectively suppress CSC pools as evidenced by decreased number of CSC marker positive cells, highlighting the suitability of this 3D culture model for evaluating CSC marker expression in a close to vivo setting. Conclusion Our results illustrate novel and previously unrecognized effects of curcumin in enhancing chemosensitization to 5-FU-based chemotherapy on DNA MMR-deficient and their chemo-resistant counterparts by targeting the CSC sub-population

Overexpression of SPARC obliterates the in vivo tumorigenicity of human hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third leading cause of cancer-related death worldwide. Current treatments are extremely disappointing. SPARC (Secreted protein, acidic and rich in cysteine) is a matricellular glycoprotein with differential expression in several tumors, including HCC, which significance remains unclear. We infected HCC cells (HepG2, Hep3B and Huh7) with an adenovirus expressing SPARC (AdsSPARC) to examine the role of SPARC expression on HCC cells and its effect on tumor aggressiveness. The in vitro HCC cells substrate-dependent proliferation and cell cycle profile were unaffected; however, SPARC overexpression reduced HCC proliferation when cells were grown in spheroids. A mild induction of cellular apoptosis was observed upon SPARC overexpression. SPARC overexpression resulted in spheroid growth inhibition in vitro while no effects were found when recombinant SPARC was exogenously applied. Moreover, the clonogenic and migratory capabilities were largely decreased in SPARC-overexpressing HCC cells, altogether suggesting a less aggressive HCC cell phenotype. Consistently, AdsSPARC-transduced cells showed increased E-cadherin expression and a concomitant decrease in N-cadherin expression. Furthermore, SPARC overexpression was found to reduce HCC cell viability in response to 5-FU-based chemotherapy in vitro, partially through induction of apoptosis. In vivo experiments revealed that SPARC overexpression in HCC cells inhibited their tumorigenic capacity and increased animal survival through a mechanism that partially involves host macrophages. Our data suggest that SPARC overexpression in HCC cells results in a reduced tumorigenicity partially through the induction of mesenchymal-to-epithelial transition (MET). These evidences point to SPARC as a novel target for HCC treatment.Fil: Atorrasagasti, María Catalina. Universidad Austral. Facultad de Ciencias Biomédicas. Laboratorio de Terapia Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Malvicini, Mariana. Universidad Austral. Facultad de Ciencias Biomédicas. Laboratorio de Terapia Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Aquino, Jorge Benjamin. Universidad Austral. Facultad de Ciencias Biomédicas. Laboratorio de Terapia Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Alaniz, Laura Daniela. Universidad Austral. Facultad de Ciencias Biomédicas. Laboratorio de Terapia Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: García, Mariana Gabriela. Universidad Austral. Facultad de Ciencias Biomédicas. Laboratorio de Terapia Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bolontrade, Marcela Fabiana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Rizzo, Manglio Miguel. Universidad Austral. Facultad de Ciencias Biomédicas. Laboratorio de Terapia Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Podhajcer, Osvaldo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Mazzolini Rizzo, Guillermo Daniel. Universidad Austral. Facultad de Ciencias Biomédicas. Laboratorio de Terapia Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Identification of Two Kinase Inhibitors with Synergistic Toxicity with Low-Dose Hydrogen Peroxide in Colorectal Cancer Cells In vitro

Colorectal carcinoma is among the most common types of cancers. With this disease, diffuse scattering in the abdominal area (peritoneal carcinosis) often occurs before diagnosis, making surgical removal of the entire malignant tissue impossible due to a large number of tumor nodules. Previous treatment options include radiation and its combination with intraperitoneal heat-induced chemotherapy (HIPEC). Both options have strong side effects and are often poor in therapeutic efficacy. Tumor cells often grow and proliferate dysregulated, with enzymes of the protein kinase family often playing a crucial role. The present study investigated whether a combination of protein kinase inhibitors and low-dose induction of oxidative stress (using hydrogen peroxide, H2O2) has an additive cytotoxic effect on murine, colorectal tumor cells (CT26). Protein kinase inhibitors from a library of 80 substances were used to investigate colorectal cancer cells for their activity, morphology, and immunogenicity (immunogenic cancer cell death, ICD) upon mono or combination. Toxic compounds identified in 2D cultures were confirmed in 3D cultures, and additive cytotoxicity was identified for the substances lavendustin A, GF109203X, and rapamycin. Toxicity was concomitant with cell cycle arrest, but except HMGB1, no increased expression of immunogenic markers was identified with the combination treatment. The results were validated for GF109203X and rapamycin but not lavendustin A in the 3D model of different colorectal (HT29, SW480) and pancreatic cancer cell lines (MiaPaca, Panc01). In conclusion, our in vitro data suggest that combining oxidative stress with chemotherapy would be conceivable to enhance antitumor efficacy in HIPEC. © 2020 by the authors. Licensee MDPI, Basel, Switzerland

PROTEOMIC IDENTIFICATION OF NOVEL MARKERS IN BREAST AND COLON CANCER

Background: Discovery of new biomarker represent the greatest promise for the detection and management of cancer. Although progress in cancer biology has been rapid during the past few years, the complete understanding of molecular basis for cancer initiation, progression and efficacious treatments is still lacking. In this context, the application of proteomic strategies is now holding a focal position. The main reason is that proteins are the functional players that drive cancer phenotypes. Among cancers, breast and colon represent the most frequent forms. The evolution of these type of cancer are not easily predictable since there are several types that behave differently among patients. The biological heterogeneity is consistent with observed varied responses to therapies across patients, also. On the other hand, drug delivery is an emergent field focused on targeting drugs to a desirable group of cells, in order to minimize undesirable side-effects and maximize the therapeutic activity. Metallic nanoparticles, in particular silver nanoparticles (Ag-NPs) exhibit low toxicity to mammalian cells (Mahapatra and Karak, 2008) and are good candidate as smart therapeutics. Based on these evidences, the first part of the study was aimed to discover new potential protein biomarkers in breast and colon cancer tissues and sera, using proteomic techniques, useful as diagnostic and prognostic factors in vivo. The second part of the study was focused on the in vitro cytotoxic effects of silver nanoparticles Ag-NPs embedded on Klebsiella Oxytoca DSM29614 (KO) Exopolysaccaride (EPS), produced in aerobic versus anaerobic conditions. Methods: Diagnostic biomarkers in breast and colon cancer: Taken advantage from previous results by the proteomic analysis performed on 13 breast cancer tissues and their matched non-tumoral adjacent tissues (Pucci-Minafra et al., 2007), we first analyze by 2D-DIGE pool of both breast and colon cancer tissues extracts compared to the matched pool of non tumoral adjacent tissues extracts. Differentially expressed proteins, identified by Maldi-TOF/TOF, were functionally clustered. We also investigate the activity levels of MMP-2 and MMP-9 in breast and colon tissues as well as in sera of the same patients. Prognostic biomarkers in breast and colon cancer: In order to identity putative proteomic signatures for colorectal cancer (CRC) metastasis, a comparative profiling of a colon cancer tissue paired with the non tumoral adjacent mucosa and with the liver metastasis from the same patient was performed. A three-step approach (normal versus tumoral versus metastasis) was used to select unique proteins involved in liver metastasis. For breast cancer, a large proteomic investigation performed on a large sample set of breast cancer patients (Cancemi et al., 2010, 2012), pointed the important role of S100 protein members in breast cancer progression. Using on line tools, for instance GOBO and breast cancer Kaplan Meir-plotter we assessed gene expression levels and clinical correlations of S100 proteins in breast patients. Cytotoxic effects of silver nanoparticles biosynthesized from KO (Ag-NPs-EPS) in SK-BR3 breast cancer cell line: We monitored cell proliferation inhibition rate by MTT assay, morphological changes and proteomic modulation. Results: Diagnostic biomarkers in breast and colon cancer: Differentially breast and bolon proteomic profiling revealed several proteins involved in common pathways among the type of cancer. The important role of MMPs in tumorigenesis was confirmed by our observations regarding their major expressions in cancer tissues compared to the normal tissues. Prognostic biomarkers in breast and colon cancer: Among the differentially expressed proteins between normal-tumor and liver metastasis, Cathepsin D expression was further analyzed as prognostic factor in CRC. Moreover, integrating results obtained by bioinformatics analysis performed on breast cancer gene expression dataset confirmed the important role of S100 proteins in breast cancer progression. Cytotoxic effects of silver nanoparticles (AgNPs) biosynthesized from KO in SK-BR3 breast cancer cell line: The most important effects were obtained by aerobically AgNPs-EPS treatment, due to the major release of Ag+1, as verified by voltammetry analysis. Morphological alteration were consistent with apoptotic features. Proteomic analysis showed modulation of several proteins related to oxidative stress and apoptotic and mitochondrial pathways. Conclusions: Conclusively, the present study contribute to the implementation of the panel of new proteomic biomarkers useful for diagnostic and prognostic applications in breast and colon cancer, providing new informations about the effects of the biosynthesized Ag-NPs-EPS on breast cancer cells

Lebanese Plants and Plant-Derived Compounds Against Colon Cancer

Colorectal cancer (CRC) is a major health concern and demands long-term efforts in developing strategies for screening and prevention. CRC has become a preventable disease as a consequence of a better understanding of colorectal carcinogenesis. However, current therapy is unsatisfactory and necessitates the exploration of other approaches for the prevention and treatment of cancer. Plant based products have been recognized as preventive with regard to the development of colon cancer. Therefore, the potential chemopreventive use and mechanism of action of Lebanese natural product were evaluated. Towards this aim the antitumor activity of Onopordum cynarocephalum and Centaurea ainetensis has been studied using in vitro and in vivo models. In vitro, both crude extracts were non cytotoxic to normal intestinal cells and inhibited the proliferation of colon cancer cells in a dose-dependent manner. In vivo, both crude extracts reduced the number of tumors by an average of 65% at weeks 20 (adenomas stage) and 30 (adenocarcinomas stage). The activity of the C. ainetensis extract was attributed to Salograviolide A, a guaianolide-type sesquiterpene lactone, which was isolated and identified through bio-guided fractionation. The mechanism of action of thymoquinone (TQ), the active component of Nigella sativa, was established in colon cancer cells using in vitro models. By the use of N-acetyl cysteine, a radical scavenger, the direct involvement of reactive oxygen species in TQ-induced apoptotic cells was established. The analytical detection of TQ from spiked serum and its protein binding were evaluated. The average recovery of TQ from spiked serum subjected to several extraction procedures was 2.5% proving the inability of conventional methods to analyze TQ from serum. This has been explained by the extensive binding (>98%) of TQ to serum and major serum components such as bovine serum albumin (BSA) and alpha-1-acid glycoprotein (AGP). Using mass spectrometry analysis, TQ was confirmed to bind covalently to the free cysteine in position 34 and 147 of the amino acid sequence of BSA and AGP, respectively. The results of this work put at the disposal for future development new plants with anti-cancer activities and enhance the understanding of the pharmaceutical properties of TQ, a prerequisite for its future clinical development.Paksusuolen syöpä on merkittävä terveyttä uhkaava sairaus ja vaatii pitkäjänteisiä ponnisteluita kehitettäessä lähestymistapoja taudin toteamiseksi ja hoitamiseksi. Paksusuolen syövän hoito on parantunut ja paksusuolen syövän kehittymiseen vaikuttavat tekijät tunnetaan paremmin kuin aikaisemmin. Kuitenkin nykyiset hoitokäytännöt eivät ole täysin tyydyttäviä ja tekevät välttämättömäksi, että tutkitaan muita tapoja ennalta ehkäistä ja hoitaa syöpää. On havaittu kasviperäisten tuotteiden estävän paksusuolen syövän kehittymistä. Tämän johdosta tässä tutkimuksessa libanonilaisten luonnontuotteiden syöpää estävää vaikutusta ja vaikutusmekanismia tutkittiin. Tutkittiin Onopordum cynarocephalum ja Centaurea ainetensis kasvien vaikutuksia paksusuolen syöpään käyttämällä in vitro and in vivo solu- ja eläinkoe malleja. In vitro, solukokeissa molemmat raakauutteet eivät olleet sytotoksisia suolistosoluille ja estivät paksusuolen syöpäsolujen lisääntymistä annosvasteisesti. In vivo, eläinkokeissa molemmat raakauutteet vähensivät kasvaimien määrää keskimäärin 65%:lla 20 viikkoa hoidon aloittamisesta (adenooma vaihe) and 30 viikkoa hoidon aloittamisesta (adenokarsinooma vaihe). C. ainetensis-uutteen aktiivisuus voitiin liittää Salograviolidi A:han, guaianolidi-tyyppinen seskviterpeenilaktoniin, mikä eristettiin ja tunnistettiin aktiivisuuden ohjaaman eristyksen avulla. Nigella sativa-kasvin vaikuttavan aineen tymokinonin (TQ) vaikutusmekanismia tutkittiin käyttämällä paksusuolen syöpäsolumalleja in vitro. Käyttämälllä N-asetyylikysteiiniä, radikaalin sieppaajaa, reaktiivisen happimolekyylien suora osallistuminen TQ-indusoiduissa apoptoottisissa soluissa voitiin osoittaa. TQ:n mittaamista seerumista ja TQ:n sitoutumista proteiineihin tutkittiin. Kun TQ oli lisätty seerumiin sen keskimääräinen saanto useiden uuttojen jälkeen oli vain 2.5%, mikä osoitti tavanomaisten menetelmien heikkouden analysoitaessa TQ:ta seerumista. Tämä selittyi TQ:n voimakkaalla sitoutumisella (>98%) seerumiin ja seerumin pääkomponentteihin kuten naudan seerumin albumiini (BSA) ja hapan glykoproteiini (alpha-1-acid glycoprotein, AGP). Käyttämällä massaspektrometri-analytiikkaa TQ:n voitiin osoittaa sitoutuvan kovalenttisesti vapaaseen kysteiiniin aminohappoissa 34 (BSA) ja 147 (AGP). Tämän työn tulokset mahdollistavat tulevaisuudessa uusien kasviperäisten syöpälääkkeiden kehittämisen ja edesauttavat TQ:n farmaseuttisten ominaisuuksien ymmärtämistä kehitettäessä sitä tulevaisuudessa lääkkeeksi kliiniseen käyttöön

Identification of two kinase inhibitors with synergistic toxicity with low-dose hydrogen peroxide in colorectal cancer cells in vitro

Colorectal carcinoma is among the most common types of cancers. With this disease, diffuse scattering in the abdominal area (peritoneal carcinosis) often occurs before diagnosis, making surgical removal of the entire malignant tissue impossible due to a large number of tumor nodules. Previous treatment options include radiation and its combination with intraperitoneal heat-induced chemotherapy (HIPEC). Both options have strong side effects and are often poor in therapeutic efficacy. Tumor cells often grow and proliferate dysregulated, with enzymes of the protein kinase family often playing a crucial role. The present study investigated whether a combination of protein kinase inhibitors and low-dose induction of oxidative stress (using hydrogen peroxide, H2O2) has an additive cytotoxic effect on murine, colorectal tumor cells (CT26). Protein kinase inhibitors from a library of 80 substances were used to investigate colorectal cancer cells for their activity, morphology, and immunogenicity (immunogenic cancer cell death, ICD) upon mono or combination. Toxic compounds identified in 2D cultures were confirmed in 3D cultures, and additive cytotoxicity was identified for the substances lavendustin A, GF109203X, and rapamycin. Toxicity was concomitant with cell cycle arrest, but except HMGB1, no increased expression of immunogenic markers was identified with the combination treatment. The results were validated for GF109203X and rapamycin but not lavendustin A in the 3D model of different colorectal (HT29, SW480) and pancreatic cancer cell lines (MiaPaca, Panc01). In conclusion, our in vitro data suggest that combining oxidative stress with chemotherapy would be conceivable to enhance antitumor efficacy in HIPEC. © 2020 by the authors. Licensee MDPI, Basel, Switzerland

DNMT3B in vitro knocking-down is able to reverse embryonal rhabdomyosarcoma cell phenotype through inhibition of proliferation and induction of myogenic differentiation

Aberrant DNA methylation has been frequently observed in many human cancers, including rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children. To date, the expression and function of the de novo DNA methyltransferase (DNMT) 3B in RMS have not yet been investigated. Our study show for the first time a significant up-regulation of DNMT3B levels in 14 RMS tumour samples and 4 RMS cell lines in comparison to normal skeletal muscle. Transfection of RD and TE671 cells, two in vitro models of embryonal RMS (ERMS), with a synthetic DNMT3B siRNA decreased cell proliferation by arresting cell cycle at G1 phase, as demonstrated by the reduced expression of Cyclin B1, Cyclin D1 and Cyclin E2, and by the concomitant up-regulation of the checkpoint regulators p21 and p27. DNMT3B depletion also impaired RB phosphorylation status and decreased migratory capacity and clonogenic potential. Interestingly, DNMT3B knock-down was able to commit ERMS cells towards myogenic terminal differentiation, as confirmed by the acquisition of a myogenic-like phenotype and by the increased expression of the myogenic markers MYOD1, Myogenin and MyHC. Finally, inhibition of MEK/ERK signalling by U0126 resulted in a reduction of DNMT3B protein, giving evidence that DNMT3B is a down-stream molecule of this oncogenic pathway.Taken together, our data indicate that altered expression of DNMT3B plays a key role in ERMS development since its silencing is able to reverse cell cancer phenotype by rescuing myogenic program. Epigenetic therapy, by targeting the DNA methylation machinery, may represent a novel therapeutic strategy against RMS

Evaluation of chemotherapeutic potential of natural extracts using 3D models of colon cancer

Dissertation to obtain master degree in BiotechnologyRecently there is a growing interest in cancer treatment through the use of natural compounds. In particular, phenolic compounds and monoterpenes found in fruits and vegetables are very attractive in the prevention and chemotherapy of various types of cancer. However, many promising compounds previously tested in in vitro cell models fail to demonstrate activity when evaluated in vivo. Therefore, there is an emerging need to develop more robust and reliable cellular models for pre-clinical evaluation of new chemotherapeutic agents. The main goal of this thesis was the evaluation of the chemotherapeutic potential of natural extracts, rich in bioactive compounds, using 3D models of colon cancer. For this purpose, a 3D model of human colorectal cancer cell line HT29 was developed. By culturing HT29 cells in a stirred culture system, it was possible to obtain 3D cellular spheroids with different size diameter during culture time. It was verified phenotypic changes within the spheroid along culture, such as formation of apoptotic core and altered expression of stem and epithelial markers in different spheroid areas, which are typical features of tumor progression. After an initial screening of the antiproliferative potential of 14 natural extracts performed in a 2D model of HT29 cells, the most promising samples were selected for further analysis in the 3D model. Cherry and orange extracts showed potential anticancer effect in HT29 aggregates through the inhibition of cell proliferation, induction of apoptosis and cell cycle arrest. A decrease on the bioactive effect was verified with the increase of aggregate diameter, probably due to limited diffusion. The anticancer activity was correlated with the phytochemical composition of natural extracts. For cherry extract, perillyl alcohol was the main bioactive compound identified whereas for orange extract, compounds like nobiletin, tangeretin and sinensetin were highlighted. Results of this thesis demonstrated that natural extracts of cherry and orange contain bioactive molecules with promising application on the development of new therapies for colon cancer treatment. The use of 3D cell models is a valuable tool for the study and evaluation of the effect of new chemotherapeutic compounds

Microbial sophorolipids inhibit colorectal tumour cell growth in vitro and restore haematocrit in Apcmin+/− mice

Sophorolipids are glycolipid biosurfactants consisting of a carbohydrate sophorose head with a fatty acid tail and exist in either an acidic or lactonic form. Sophorolipids are gaining interest as potential cancer chemotherapeutics due to their inhibitory effects on a range of tumour cell lines. Currently, most anti-cancer studies reporting the effects of sophorolipids have focused on lactonic preparations with the effects of acidic sophorolipids yet to be elucidated. We produced a 94% pure acidic sophorolipid preparation which proved to be non-toxic to normal human colonic and lung cells. In contrast, we observed a dose-dependent reduction in viability of colorectal cancer lines treated with the same preparation. Acidic sophorolipids induced apoptosis and necrosis, reduced migration, and inhibited colony formation in all cancer cell lines tested. Furthermore, oral administration of 50 mg kg(-1) acidic sophorolipids over 70 days to Apc(min+/-) mice was well tolerated and resulted in an increased haematocrit, as well as reducing splenic size and red pulp area. Oral feeding did not affect tumour numbers or sizes in this model. This is the first study to show that acidic sophorolipids dose-dependently and specifically reduces colon cancer cell viability in addition to reducing tumour-associated bleeding in the Apc(min+/-) mouse model