S-Adenosylmethionine Increases the Sensitivity of Human Colorectal Cancer Cells to 5-Fluorouracil by Inhibiting P-Glycoprotein Expression and NF-κB Activation

Colorectal cancer (CRC) is the second deadliest cancer worldwide despite significant advances in both diagnosis and therapy. The high incidence of CRC and its poor prognosis, partially attributed to multi-drug resistance and antiapoptotic activity of cancer cells, arouse strong interest in the identification and development of new treatments. S-Adenosylmethionine (AdoMet), a natural compound and a nutritional supplement, is well known for its antiproliferative and proapoptotic effects as well as for its potential in overcoming drug resistance in many kinds of human tumors. Here, we report that AdoMet enhanced the antitumor activity of 5-Fluorouracil (5-FU) in HCT 116^p53+/+ and in LoVo CRC cells through the inhibition of autophagy, induced by 5-FU as a cell defense mechanism to escape the drug cytotoxicity. Multiple drug resistance is mainly due to the overexpression of drug efflux pumps, such as P-glycoprotein (P-gp). We demonstrate here that AdoMet was able to revert the 5-FU-induced upregulation of P-gp expression and to decrease levels of acetylated NF-κB, the activated form of NF-κB, the major antiapoptotic factor involved in P-gp-related chemoresistance. Overall, our data show that AdoMet, was able to overcome 5-FU chemoresistance in CRC cells by targeting multiple pathways such as autophagy, P-gp expression, and NF-κB signaling activation and provided important implications for the development of new adjuvant therapies to improve CRC treatment and patient outcomes.     

Prunus spinosa Extract Sensitized HCT116 Spheroids to 5-Fluorouracil Toxicity,Inhibiting Autophagy

Autophagy is a lysosomal degradation and recycling process involved in tumor progression and drug resistance. The aim of this work was to inhibit autophagy and increase apoptosis in a 3D model of human colorectal cancer by combined treatment with our patented natural product Prunus spinosa + nutraceutical activator complex (PsT + NAC^®) and 5-fluorouracil (5-FU). By means of cytotoxic evaluation (MTT assay), cytofluorimetric analysis, light and fluorescence microscopy investigation and Western blotting evaluation of the molecular pathway PI3/AKT/mTOR, Caspase-9, Caspase-3, Beclin1, p62 and LC3, we demonstrated that the combination PsT + NAC^® and 5-FU significantly reduces autophagy by increasing the apoptotic phenomenon. These results demonstrate the importance of using non-toxic natural compounds to improve the therapeutic efficacy and reduce the side effects induced by conventional drugs in human colon cancer.     

Molecular Effectors of Photodynamic Therapy-Mediated Resistance to Cancer Cells

Photodynamic therapy (PDT) is currently enjoying considerable attention as the subject of experimental research to treat resistant cancers. The preferential accumulation of a non-toxic photosensitizer (PS) in different cellular organelles that causes oxidative damage by combining light and molecular oxygen leads to selective cell killing. However, one major setback, common among other treatment approaches, is tumor relapse and the development of resistance causing treatment failure. PDT-mediated resistance could result from increased drug efflux and decreased localization of PS, reduced light exposure, increased DNA damage repair, and altered expression of survival genes. This review highlights the essential insights of PDT reports in which PDT resistance was observed and which identified some of the molecular effectors that facilitate the development of PDT resistance. We also discuss different perceptions of PDT and how its current limitations can be overturned to design improved cancer resistant treatments.     

Kaempferol Can Reverse the 5-Fu Resistance of Colorectal Cancer Cells by Inhibiting PKM2-Mediated Glycolysis

Resistance to 5-Fluorouracil (5-Fu) chemotherapy is the main cause of treatment failure in the cure of colon cancer. Therefore, there is an urgent need to explore a safe and effective multidrug resistance reversal agent for colorectal cancer, which would be of great significance for improving clinical efficacy. The dietary flavonoid kaempferol plays a key role in the progression of colorectal cancer and 5-Fu resistance. However, the molecular mechanism of kaempferol in reversing 5-Fu resistance in human colorectal cancer cells is still unclear. We found that kaempferol could reverse the drug resistance of HCT8-R cells to 5-Fu, suggesting that kaempferol alone or in combination with 5-Fu has the potential to treat colorectal cancer. It is well known that aerobic glycolysis is related to tumor growth and chemotherapy resistance. Indeed, kaempferol treatment significantly reduced glucose uptake and lactic acid production in drug-resistant colorectal cancer cells. In terms of mechanism, kaempferol promotes the expression of microRNA-326 (miR-326) in colon cancer cells, and miR-326 could inhibit the process of glycolysis by directly targeting pyruvate kinase M2 isoform (PKM2) 3′-UTR (untranslated region) to inhibit the expression of PKM2 or indirectly block the alternative splicing factors of PKM mRNA, and then reverse the resistance of colorectal cancer cells to 5-Fu. Taken together, our data suggest that kaempferol may play an important role in overcoming resistance to 5-Fu therapy by regulating the miR-326-hnRNPA1/A2/PTBP1-PKM2 axis.     

Molecular Mechanisms of Tumor Immunomodulation in the Microenvironment of Colorectal Cancer

Colorectal cancer remains one of the most important health challenges in our society. The development of cancer immunotherapies has fostered the need to better understand the anti-tumor immune mechanisms at play in the tumor microenvironment and the strategies by which the tumor escapes them. In this review, we provide an overview of the molecular interactions that regulate tumor inflammation. We particularly discuss immunomodulatory cell-cell interactions, cell-soluble factor interactions, cell-extracellular matrix interactions and cell-microbiome interactions. While doing so, we highlight relevant examples of tumor immunomodulation in colorectal cancer.     

Structural Properties of Polyphenols Causing Cell Cycle Arrest at G1 Phase in HCT116 Human Colorectal Cancer Cell Lines

Plant-derived polyphenols are being tested as chemopreventive agents; some polyphenols arrest the cell cycle at G1 phase, whereas others inhibit cell cycle proliferation at G2/M phase. Therefore, polyphenols have been proposed to inhibit cell cycle progression at different phases via distinct mechanisms. Indeed, our previous studies showed that small structural differences in polyphenols cause large differences in their biological activities; however, the details of the structural properties causing G1 cell cycle arrest remain unknown. In this study, we prepared 27 polyphenols, including eight different scaffolds, to gain insight into the structural conditions that arrest the cell cycle at G1 phase in a quantitative structure–activity relationship study. We used cell cycle profiles to determine the biophores responsible for G1 cell cycle arrest and believe that the biophores identified in this study will help design polyphenols that cause G1 cell cycle arrest.     

Brain Metastases from Colorectal Cancer: Microenvironment and Molecular Mechanisms

Colorectal cancer is one of the most common digestive tract malignancies in the world. Owing to the newer and more effective systemic therapies, the life of colorectal cancer patients can be remarkably prolonged, and the incidence of brain metastases is increasing. However, little is known about the underlying mechanisms of brain metastasis from colorectal cancer. Here we review the tumor microenvironment and metastasis associated molecules in brain metastases from colorectal cancer. A further understanding of these mechanisms will help us to propose better strategies for colorectal cancer patients with brain metastasis and improve their life quality.     

The Role of Tumor Microenvironment Cells in Colorectal Cancer (CRC) Cachexia

Cancer cachexia (CC) is a multifactorial syndrome in patients with advanced cancer characterized by weight loss via skeletal-muscle and adipose-tissue atrophy, catabolic activity, and systemic inflammation. CC is correlated with functional impairment, reduced therapeutic responsiveness, and poor prognosis, and is a major cause of death in cancer patients. In colorectal cancer (CRC), cachexia affects around 50–61% of patients, but remains overlooked, understudied, and uncured. The mechanisms driving CC are not fully understood but are related, at least in part, to the local and systemic immune response to the tumor. Accumulating evidence demonstrates a significant role of tumor microenvironment (TME) cells (e.g., macrophages, neutrophils, and fibroblasts) in both cancer progression and tumor-induced cachexia, through the production of multiple procachectic factors. The most important role in CRC-associated cachexia is played by pro-inflammatory cytokines, including the tumor necrosis factor α (TNFα), originally known as cachectin, Interleukin (IL)-1, IL-6, and certain chemokines (e.g., IL-8). Heterogeneous CRC cells themselves also produce numerous cytokines (including chemokines), as well as novel factors called “cachexokines”. The tumor microenvironment (TME) contributes to systemic inflammation and increased oxidative stress and fibrosis. This review summarizes the current knowledge on the role of TME cellular components in CRC-associated cachexia, as well as discusses the potential role of selected mediators secreted by colorectal cancer cells in cooperation with tumor-associated immune and non-immune cells of tumor microenvironment in inducing or potentiating cancer cachexia. This knowledge serves to aid the understanding of the mechanisms of this process, as well as prevent its consequences.     

The Role of Exosomal Non-Coding RNAs in Colorectal Cancer Drug Resistance

Background: Colorectal cancer (CRC) is one of the most common types of cancer diagnosed worldwide with high morbidity; drug resistance is often responsible for treatment failure in CRC. Non-coding RNAs (ncRNAs) play distinct regulatory roles in tumorigenesis, cancer progression and chemoresistance. Methods: A literature search was conducted in PubMed database in order to sum up and discuss the role of exosomal ncRNAs (ex-ncRNAs) in CRC drug resistance/response and their possible mechanisms. Results: Thirty-six (36) original research articles were identified; these included exosome or extracellular vesicle (EV)-containing microRNAs (miRNAs), long non-coding RNAs (lncRNAs), circular RNAs (circRNAs) and small-interfering (siRNAs). No studies were found for piwi-interacting RNAs. Conclusions: Exosomal transfer of ncRNAs has been documented as a new mechanism of CRC drug resistance. Despite being in its infancy, it has emerged as a promising field for research in order to (i) discover novel biomarkers for therapy monitoring and/or (ii) reverse drug desensitization.     

Repurposing Alone and in Combination of the Antiviral Saquinavir with 5-Fluorouracil in Prostate and Lung Cancer Cells

Prostate and lung cancers are among the most common cancer types, and they still need more therapeutics. For this purpose, saquinavir (SAQ) was tested alone and in combination with 5-fluorouracil (5-FU). PC-3 and A549 cells were exposed to increasing concentrations of both drugs alone or in combination, with simultaneous or sequential administration. Cell viability was obtained using the MTT assay and synergism values using CompuSyn software. Results showed that SAQ was the more cytotoxic of both drugs in PC-3 cells, while 5-FU was the most cytotoxic in A549 cells. When these drugs were used in combination, the more synergistic combination in PC-3 cells was the IC₅₀ of SAQ with various concentrations of 5-FU, particularly when 5-FU was only applied 24 h later. Meanwhile for A549 the most promising combination was 5-FU with delayed SAQ, but with a weaker effect than all combinations demonstrated in PC-3 cells. These results demonstrate that SAQ could be used as a new repurposed drug for the treatment of prostate cancer and this treatment potential could be even greater if SAQ is combined with the anticancer drug 5-FU, while for lung cancer it is not as efficient and, therefore, not of as much interest.     

MicroRNA-19b Plays a Key Role in 5-Fluorouracil Resistance and Predicts Tumor Progression in Locally Advanced Rectal Cancer Patients

The standard clinical management of locally advanced rectal cancer (LARC) patients includes neoadjuvant 5-fluorouracil (5-FU)-based chemoradiotherapy (CRT) followed by mesorectal excision. MicroRNA (miR)-19b expression levels in LARC biopsies obtained from initial colonoscopy have recently been identified as independent predictors of both patient outcome and pathological response to preoperative CRT in this disease. Moreover, it has been discovered that this miR increases its expression in 5-FU resistant colon cancer cells after 5-FU exposure. Despite the fact that these observations suggest a functional role of miR-19b modulating 5-FU response of LARC cells, this issue still remains to be clarified. Here, we show that downregulation of miR-19b enhances the antitumor effects of 5-FU treatment. Moreover, ectopic miR-19b modulation was able to restore sensitivity to 5-FU treatment using an acquired resistant model to this compound. Notably, we also evaluated the potential clinical impact of miR-19b as a predictive marker of disease progression after tumor surgery resection in LARC patients, observing that miR-19b overexpression significantly anticipates patient recurrence in our cohort (p = 0.002). Altogether, our findings demonstrate the functional role of miR-19b in the progressively decreasing sensitivity to 5-FU treatment and its potential usefulness as a therapeutic target to overcome 5-FU resistance, as well as its clinical impact as predictor of tumor progression and relapse.     

EPLIN,a Putative Tumour Suppressor in Colorectal Cancer,Implications in Drug Resistance

Colorectal cancer is a serious threat to human health. Poor prognosis and frequently reported drug resistance urges research into novel biomarkers and mechanisms to aid in the understanding of the development and progression of colorectal cancer and to optimise therapeutic strategies. In the current study, we investigated the roles of a putative tumour suppressor, EPLIN, in colorectal cancer. Our clinical colorectal cancer cohort and online databases revealed a downregulation of EPLIN in colorectal cancer tissues compared with normal tissues. The reduced expression of EPLIN was associated with poor clinical outcomes of patients. In vitro cellular function assays showed that EPLIN elicited an inhibitory effect on cellular growth, adhesion, migration and invasion. Utilising a protein microarray on protein samples from normal and tumour patient tissues suggested HSP60, Her2 and other signalling events were novel potential interacting partners of EPLIN. It was further revealed that EPLIN and HSP60 were negative regulators of Her2 in colorectal cancer cells. The clinical cohort also demonstrated that expression of HSP60 and Her2 affected clinical outcomes, but most interestingly the combination of EPLIN, HSP60 and Her2 was able to identify patients with the most unfavourable clinical outcome by independently predicting patient overall survival and disease free survival. Furthermore, EPLIN and HSP60 exhibited potential to regulate cellular response to chemotherapeutic and EGFR/Her2 targeted therapeutic agents. In conclusion, EPLIN is an important prognostic factor for patients with colon cancer and reduced EPLIN in CRC contributes to aggressive traits of CRC cells and their responses to chemotherapeutic drugs. Collectively, EPLIN is a pivotal factor for the development and progression of colorectal cancer and has important clinical and therapeutic values in this cancer type.     

Piperine Targets Different Drug Resistance Mechanisms in Human Ovarian Cancer Cell Lines Leading to Increased Sensitivity to Cytotoxic Drugs

Our goal was to examine the anticancer effects of piperine against the resistant human ovarian cancer cells and to explore the molecular mechanisms responsible for its anticancer effects. Our study used drug-sensitive ovarian cancer cell line W1 and its sublines resistant to paclitaxel (PAC) and topotecan (TOP). We analyzed the cytotoxic effect of piperine and cytostatic drugs using an MTT assay. The impact of piperine on protein expression was determined by immunofluorescence and Western blot. We also examined its effect on cell proliferation and migration. We noticed a different level of piperine resistance between cell lines. Piperine increases the cytotoxic effect of PAC and TOP in drug-resistant cells. We observed an increase in PTPRK expression correlated with decreased pTYR level after piperine treatment and downregulation of P-gp and BCRP expression. We also noted a decrease in COL3A1 and TGFBI expression in investigated cell lines and increased COL3A1 expression in media from W1PR2 cells. The expression of Ki67 protein and cell proliferation rate decreased after piperine treatment. Piperine markedly inhibited W1TR cell migration. Piperine can be considered a potential anticancer agent that can increase chemotherapy effectiveness in cancer patients.     

Circulating Tumor Cells in Colorectal Cancer: Detection Systems and Clinical Utility

Colorectal cancer (CRC) is the third most common cancer worldwide. The high mortality from CRC is mainly related to metastasis affecting distant organs and their function. Dissemination of tumor cells from the primary tumor and hematogeneous spread are considered crucial in the formation of tumor metastases. The analysis of circulating tumor cells (CTCs) and CTC clusters in the blood can be used for the early detection of invasive cancer. Moreover, CTCs have a prognostic significance in the monitoring of a malignant disease or the response to chemotherapy. This work presents an overview of the research conducted on CTCs with the aim of finding suitable detection systems and assessing the possibility of clinical applications in patients with CRC.     

Portrait of Cancer Stem Cells on Colorectal Cancer: Molecular Biomarkers,Signaling Pathways and miRNAome

Colorectal cancer (CRC) is a leading cause of cancer death worldwide, and about 20% is metastatic at diagnosis and untreatable. Increasing evidence suggests that the heterogeneous nature of CRC is related to colorectal cancer stem cells (CCSCs), a small cells population with stemness behaviors and responsible for tumor progression, recurrence, and therapy resistance. Growing knowledge of stem cells (SCs) biology has rapidly improved uncovering the molecular mechanisms and possible crosstalk/feedback loops between signaling pathways that directly influence intestinal homeostasis and tumorigenesis. The generation of CCSCs is probably connected to genetic changes in members of signaling pathways, which control self-renewal and pluripotency in SCs and then establish function and phenotype of CCSCs. Particularly, various deregulated CCSC-related miRNAs have been reported to modulate stemness features, controlling CCSCs functions such as regulation of cell cycle genes expression, epithelial-mesenchymal transition, metastasization, and drug-resistance mechanisms. Primarily, CCSC-related miRNAs work by regulating mainly signal pathways known to be involved in CCSCs biology. This review intends to summarize the epigenetic findings linked to miRNAome in the maintenance and regulation of CCSCs, including their relationships with different signaling pathways, which should help to identify specific diagnostic, prognostic, and predictive biomarkers for CRC, but also develop innovative CCSCs-targeted therapies.     

Cadmium Modifies the Cell Cycle and Apoptotic Profiles of Human Breast Cancer Cells Treated with 5-Fluorouracil

Industrialisation, the proximity of factories to cities, and human work activities have led to a disproportionate use of substances containing heavy metals, such as cadmium (Cd), which may have deleterious effects on human health. Carcinogenic effects of Cd and its relationship with breast cancer, among other tumours, have been reported. 5-Fluorouracil (5-FU) is a fluoropyrimidine anticancer drug used to treat solid tumours of the colon, breast, stomach, liver, and pancreas. The purpose of this work was to study the effects of Cd on cell cycle, apoptosis, and gene and protein expression in MCF-7 breast cancer cells treated with 5-FU. Cd altered the cell cycle profile, and its effects were greater when used either alone or in combination with 5-FU compared with 5-FU alone. Cd significantly suppressed apoptosis of MCF-7 cells pre-treated with 5-FU. Regarding gene and protein expression, bcl2 expression was mainly upregulated by all treatments involving Cd. The expression of caspase 8 and caspase 9 was decreased by most of the treatments and at all times evaluated. C-myc expression was increased by all treatments involving Cd, especially 5-FU plus Cd at the half time of treatment. Cd plus 5-FU decreased cyclin D1 and increased cyclin A1 expression. In conclusion, our results indicate that exposure to Cd blocks the anticancer effects of 5-FU in MCF-7 cells. These results could have important clinical implications in patients treated with 5-FU-based therapies and who are exposed to high levels of Cd.     

Extracellular Disposal of Tumor-Suppressor miRs-145 and -34a via Microvesicles and 5-FU Resistance of Human Colon Cancer Cells

The dysregulation of microRNA (miRNA) expression causes various kinds of diseases. Especially, alterations in miRNA expression levels are frequently observed in human tumor cells and are associated with cancer pathogenesis. Earlier we established Fluorouracil (5-FU)-resistant human colon cancer DLD-1 cells (DLD-1/5FU) from parental 5-FU- sensitive DLD-1 cells. In the present study, we examined the expression of miRNA in each cell line and in its extracellular microvesicles (MVs) before and after treatment with 5-FU. The nascent RNAs of anti-oncogenic miR-34a and -145 labeled with EU in both cells were proved to be transferred into MVs in both cell lines. The levels of miR-34a and -145 in the cells and in their MVs were not largely different in the two cell lines, and a substantial amount of both miRNAs was secreted by both cell lines even in the steady-state condition. The exposure of both cell lines to 5-FU significantly increased the intracellular levels of miR-145 and miR-34a in the 5-FU-sensitive DLD-1 cells, whereas the level of neither miR was elevated in the DLD-1/5FU cells. Interestingly, the amount of miR-145 detected in the small MVs shed into the medium of the parental cells was reduced after the treatment with 5-FU. On the other hand, the intracellular expression of miR-34a in the DLD-1/5FU cells was down-regulated compared with that in the parental DLD-1 cells even in the steady-state condition. As to the miR-34a secreted into MVs, the increase in the level in DLD-1/5FU cells was greater than that in the parental DLD-1 cells after the treatment with 5-FU. Thus, the intra- and extracellular miR-145 and -34a were closely associated with 5-FU resistance, and the resistance was in part due to the enhanced secretion of miR-145 and -34a via MVs, resulting in low intracellular levels of both miRNAs.     

Coprinopsis cinerea Galectin CGL1 Induces Apoptosis and Inhibits Tumor Growth in Colorectal Cancer Cells

Mushroom galectins are promising anticancer agents for their low IC₅₀ values against cancer cells in vitro. In this study, two Coprinopsis cinerea galectins, CGL1 and CGL2, were heterologously expressed, and their biochemistry properties and anticancer effects were evaluated. The purified galectins were thermostable at neutral pH conditions. They both existed as tetramers and shared a high affinity towards lactose. CGL1 and CGL2 strongly inhibited the cell viability of many cancer cell lines, including three colorectal cancer cells, in a dose-dependent manner by inducing mitochondria-mediated caspase-dependent apoptosis. Furthermore, CGL1 exhibited higher apoptosis-inducing ability and cytotoxicity than CGL2. In vivo cell viability experiments based on two xenograft mouse models showed that CGL1 had a more substantial inhibitory effect than CGL2 on HCT116 tumor growth (p < 0.0001), whereas only CGL1 inhibited DLD1 tumor growth (p < 0.01). This is the first study to evaluate the anti-colorectal cancer effect of mushroom lectins in vivo, and our results showed that CGL1 is a potent agent for colorectal cancer treatment.     

Defective Generation and Maturation of Dendritic Cells from Monocytes in Colorectal Cancer Patients during the Course of Disease

Colorectal cancer (CRC) is the second-leading cause of cancer-related deaths in Western countries. Today, the role of the host’s immune system in controlling the progression and spread of solid tumors is broadly established. Tumor immunosurveillance escape mechanisms, such as those involving dendritic cells (DCs), the most important antigen-presenting cells, are likewise recognized processes involved in cancer. The present study evaluates the ability of CRC patients to generate DCs in vitro from circulating monocytes at both pre- and post-operative timepoints; the results are correlated with the stage of disease to shed light on the systemic immune statuses of CRC patients. Our data showed that patients’ DCs had lower co-stimulatory molecule expression and were less able to present antigens to allogeneic T cells compared to healthy controls’ (HC) DCs. Furthermore altered cytokine secretion, such as increased IL-10 and reduced IL-12 and TNF-α, was observed. At the post-operative timepoints we observed a recovery of the patients’ ability to generate immature DCs, compared to HCs, but the maturational capacity remained affected. Our study conclusively highlights the persistently impaired in vitro generation of fully mature and functional DCs, which appears to be more altered during advanced stages. This work sheds light on a dendritic cell-based tumor immune escape mechanism that could be useful for the development of more effective immunotherapeutic strategies.