Survival Of Colon Cancer Cells Research Articles

LncRNA HAND2-AS1 Inhibited Colon Cancer Progression By Regulating miR-3118/ZG16 Axis.

LncRNA HAND2-AS1 is a novel cancer regulator, but the role and mechanisms of HAND2-AS1 involved with colon cancer (CC) progression remains unknown. The purpose of this research was to figure out how HAND2-AS1 regulates the progression of CC. Using qRT-PCR, we studied expression levels of miR-3118, HAND2-AS1, and ZG16 in CC tissues and cells. Protein levels of apoptosis-related proteins (Bax and Bcl-2) and ZG16 were quantified by western blotting. In vitro function analysis referred to western blotting, wound healing assay and CCK-8. The binding association among miR-3118, HAND2-AS1, and ZG16 was investigated using luciferase reporter and RIP assays. The functional role of HAND2-AS1 was analyzed using xenograft tumor models in vivo. In tissues and cells of CC, HAND2-AS1 was downregulated. We observed that HAND2-AS1 overexpression declined CC cell proliferation and migration while facilitating apoptosis. We further verified that when HAND2-AS1 is overexpressed it reduced CC tumor development in vivo. In CC cells and tissues, miR-3118 competed with HAND2-AS1 and was elevated. Further it was noted that the HAND2-AS1 when overexpressed, lessened the survival of CC cells, however overexpression of miR-3118 restored these changes. ZG16 was shown to be a target of miR-3118, it was found that ZG16 was downregulated in CC tissue and cells. We observed, high expression of ZG16 partially restored the enhanced malignant phenotype caused by miR-3118 overexpression. HAND2-AS1 inhibited CC progression by upregulating ZG16 expression through sponging miR-3118. Hence, HAND2-AS1/miR-3118/ZG16 axis could be a possible new target for CC treatment.

RIOK3 sustains colorectal cancer cell survival under glucose deprivation via an HSP90α-dependent pathway

Glucose oxidation via the pentose phosphate pathway serves as the primary cellular mechanism for generating nicotinamide adenine dinucleotide phosphate (NADPH). The central regions of solid tumors typically experience glucose deficiency, emphasizing the need for sustained NADPH production crucial to tumor cell survival. This study highlights the crucial role of RIOK3 in maintaining NADPH production and colorectal cancer (CRC) cell survival during glucose deficiency. Our findings revealed upregulated RIOK3 expression upon glucose deprivation, with RIOK3 knockout significantly reducing cancer cell survival. Mechanistically, RIOK3 interacts with heat shock protein 90α (HSP90α), a chaperone integral to various cellular processes, thereby facilitating HSP90α binding to isocitrate dehydrogenase 1 (IDH1). This interaction further upregulates IDH1 expression, enhancing NADPH production and preserving redox balance. Furthermore, RIOK3 inhibition had no discernible effect on intracellular NADPH levels and cell death rates in HSP90α-knockdown cells. Collectively, our findings suggest that RIOK3 sustains colon cancer cell survival in low-glucose environments through an HSP90α-dependent pathway. This highlights the significance of the RIOK3–HSP90α–IDH1 cascade, providing insights into potential targeted therapeutic strategies for CRC in metabolic stress conditions.

Inhibition of ubiquitin specific peptidase 8 is effective against 5-fluorouracil resistance in colon cancer via suppressing EGFR and EGFR-mediated signaling pathways.

The identification of a sensitizing strategy to overcome 5-fluorouracil (5-FU) therapeutic resistance is needed in colon cancer. Recent studies highlight the oncogenic role of ubiquitin specific peptidase 8 (USP8) in many cancers. In line with these efforts, this work investigated the therapeutic potential of targeting USP8 in colon cancer. Immunohistochemistry was performed to determine USP8 expression level in colon cancer tissues and their adjacent normal tissues. Gain-of-function analysis via plasmid overexpression and loss-of-function analysis via siRNA knockdown were applied on cellular assays. The combinatory effects of USP8 inhibitor and cisplatin were determined using a colon xenograft mouse model. Immunoblotting was performed to investigate the molecular mechanism of USP8 inhibition in colon cancer cells. Compared to normal counterparts, we showed that USP8 protein level was significantly higher in colon cancer tissues and cells. In addition, USP8 expression was not affected by prolonged exposure of colon cancer cells to 5-FU. USP8 was important for colon cancer cell growth and survival but not migration as assessed by loss-of-function and gain-of-function approaches. Pharmacological inhibition of USP8 using USP8 inhibitor is active against both sensitive and 5-FU-resistant colon cancer cells. Of note, USP8 inhibitor significantly inhibited colon cancer formation and growth, and augmented in vivo efficacy of 5-FU without causing toxicity in mice. Mechanistic studies showed that USP8 inhibitor acted on colon cancer cells through suppressing EGFR and EGFR-mediated signalling pathways. Our work is the first to reveal the essential role of USP8 in colon cancer via EGFR oncogenic signalling pathways. Our findings provide a proof-of-concept that USP8 inhibitors are promising candidates to overcome 5-FU resistance in colon cancer.

In-vitro Studies of Extracts of Plumbago Zeylanica in Cancer Cell Lines.

Despite increased use of early detection methods and more aggressive treatment strategies, the worldwide incidence of colorectal cancer is still on the rise. Consequently, it remains urgent to identify novel agents with enhanced efficacy in prevention and/or therapeutic protocols. Our studies focused on the use of Plumbagin, a natural phytochemical that showed promising results against other tumor types, to determine its effectiveness in blocking the proliferation and survival of colon cancer cells in experimental protocols mimicking the environment in primary tumors (attached culture conditions) and in circulating tumor cells (unattached conditions). Under both experimental settings, exposure of HCT116 cells to Plumbagin concentrations in the low micromolar range resulted in cell cycle arrest at the G1 phase, apoptosis via the mitochondrial cell death pathway, and increased production of reactive oxygen species. The cell cycle effects were more noticeable in attached cells, whereas the induction of cell death was more evident in unattached cells. These effects were consistent with the nature and the magnitude of the alterations induced by Plumbagin on the expression levels of a set of proteins known to play key roles in the regulation of cell cycle dynamics, apoptosis mechanisms and cell proliferation. In light of its previously reported lack of toxicity on normal colon cells and the striking anti-survival effect on colon cancer cells observed in our study, Plumbagin should be considered a promising drug for the treatment of colon cancer.

Inhibition of mitochondrial fission activates glycogen synthesis to support cell survival in colon cancer

Metabolic reprogramming has been recognized as one of the major mechanisms that fuel tumor initiation and progression. Our previous studies demonstrate that activation of Drp1 promotes fatty acid oxidation and downstream Wnt signaling. Here we investigate the role of Drp1 in regulating glycogen metabolism in colon cancer. Knockdown of Drp1 decreases mitochondrial respiration without increasing glycolysis. Analysis of cellular metabolites reveals that the levels of glucose-6-phosphate, a precursor for glycogenesis, are significantly elevated whereas pyruvate and other TCA cycle metabolites remain unchanged in Drp1 knockdown cells. Additionally, silencing Drp1 activates AMPK to stimulate the expression glycogen synthase 1 (GYS1) mRNA and promote glycogen storage. Using 3D organoids from Apcf/f/Villin-CreERT2 models, we show that glycogen levels are elevated in tumor organoids upon genetic deletion of Drp1. Similarly, increased GYS1 expression and glycogen accumulation are detected in xenograft tumors derived from Drp1 knockdown colon cancer cells. Functionally, increased glycogen storage provides survival advantage to Drp1 knockdown cells. Co-targeting glycogen phosphorylase-mediated glycogenolysis sensitizes Drp1 knockdown cells to chemotherapy drug treatment. Taken together, our results suggest that Drp1-loss activates glucose uptake and glycogenesis as compensative metabolic pathways to promote cell survival. Combined inhibition of glycogen metabolism may enhance the efficacy of chemotherapeutic agents for colon cancer treatment.

Triggering of Endoplasmic Reticulum Stress by Tannic Acid Inhibits the Proliferation and Migration of Colorectal Cancer Cells.

Due to the pivotal role of endoplasmic reticulum (ER) stress in cancers, interfering with its function can cause the accumulation of unfolded proteins, which ultimately leads to the activation of the unfolded protein response (UPR) signaling pathway and apoptosis. Therefore, the use of plant compounds such as tannic acid with UPR-inducing properties can be proposed as a possible treatment method for cancer. In this study, we investigated the effect of tannic acid on cell migration, colony formation, growth, and UPR-induced apoptosis in the SW48 colorectal cancer cell line. The MTT assay was performed to investigate the cytotoxic effect of tannic acid. We performed the qPCR method to elucidate the effect of tannic acid on the expression of Bim, MMP-9, Bcl-xL, cyclin D1, CHOP, and ATF4 genes. We also used the colony formation and migration experiments to investigate the effect of this compound on the colony formation and migration ability of tumor cells. Finally, we used Hoechst staining to measure cell apoptosis. Tannic acid inhibited the cell survival, clonogenic, and migration of colon cancer cells. This compound increased the expression of ER stress-mediated UPR genes, ATF4 and CHOP. Moreover; tannic acid increased the expression of pro-apoptotic proteins like Bim, while at the same time causing a sharp decline in the expression of anti-apoptotic protein Bcl-xL. A decline in MMP-9 expression confirmed the anti-metastatic role of this compound. Taken together, tannic acid can induce apoptosis via ER stress-mediated UPR pathway, and has a suppressive effect on cell viability, growth, migration, colony formation, and metastasis, suggesting it may be a potential drug in colorectal cancer treatment.

Abstract 4837: Inhibition of mitochondrial fission activates glycogen storage to support cell survival in colon cancer

Abstract Metabolic reprogramming has been increasingly recognized as one of the major mechanisms that fuels tumorigenesis and disease progression. Our previous studies have shown that in response to fatty acid uptake, colon cancer cells activate mitochondrial fission to support fatty acid oxidation and downstream Wnt signaling. Given the potential benefit of inhibiting mitochondrial fission, Dynamin-Related Protein 1 (Drp1), a pro-fission factor, has become an attractive target for developing anticancer agents. Here we investigated the role of Drp1 in promoting metabolic adaptation in colon cancer. We found that knockdown of Drp1 decreased mitochondrial respiration which resulted in increased glucose uptake and lactate production. In addition, downregulation of Drp1 increased AMP-activated protein kinase (AMPK) activity which coincided with glycogen accumulation. Consistently, results from GC/MS analysis of cellular metabolites revealed that the levels of glucose-6-phosphate, a precursor for glycogenesis, were significantly elevated in Drp1 knockdown cells whereas pyruvate and other TCA cycle metabolites remained unchanged. Mechanistically, AMPK transcriptionally activates the expression of glycogen synthase 1 (GYS1) and hexokinase 2 (HK2) genes and silencing GYS1 abolished the glycogen accumulation phenotype in Drp1 knockdown cells. Using APC-derived 3D organoids, we demonstrated that the glycogen levels were elevated in Apcf/f Drp1f/f tumor organoids upon deletion of Drp1. Similarly, increased GYS1 expression and glycogen levels were detected in xenograft tumors derived from Drp1 knockdown colon cancer cells compared to control. Functionally, increased glycogen storage allowed Drp1 knockdown cells to survive glucose starvation conditions suggesting an enhanced survival capacity compared to control cells. Taken together, our findings indicate that Drp1 inhibition by itself is unlikely to be sufficient to eradicate cancer cells as adaptive metabolic mechanisms are activated to promote cell survival. However, combining Drp1 inhibition may enhance the efficacy of chemotherapeutic agents for colon cancer treatment. Citation Format: Sumati Hasani, Lyndsay E. Young, Moumita Banerjee, Dylan Rivas, Jinhwan Kim, Ramon Sun, Matthew Gentry, Xiaopeng Xiong, Tianyan Gao. Inhibition of mitochondrial fission activates glycogen storage to support cell survival in colon cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4837.

5-Fluorouracil Suppresses Colon Tumor through Activating the p53-Fas Pathway to Sensitize Myeloid-Derived Suppressor Cells to FasL+ Cytotoxic T Lymphocyte Cytotoxicity.

Myelosuppression is a major adverse effect of 5-fluorouracil (5-FU) chemotherapy. However, recent findings indicate that 5-FU selectively suppresses myeloid-derived suppressor cells (MDSCs), to enhance antitumor immunity in tumor-bearing mice. 5-FU-mediated myelosuppression may thus have a beneficial effect for cancer patients. The molecular mechanism underlying 5-FU's suppression of MDSCs is currently unknown. We aimed at testing the hypothesis that 5-FU suppresses MDSCs through enhancing MDSC sensitivity to Fas-mediated apoptosis. We observed that, although FasL is highly expressed in T cells, Fas is weakly expressed in myeloid cells in human colon carcinoma, indicating that downregulation of Fas is a mechanism underlying myeloid cell survival and accumulation in human colon cancer. 5-FU treatment upregulated expression of both p53 and Fas, and knocking down p53 diminished 5-FU-induced Fas expression in MDSC-like cells, in vitro. 5-FU treatment also increased MDSC-like cell sensitivity to FasL-induced apoptosis in vitro. Furthermore, we determined that 5-FU therapy increased expression of Fas on MDSCs, suppressed MDSC accumulation, and increased CTL tumor infiltration in colon tumor-bearing mice. In human colorectal cancer patients, 5-FU chemotherapy decreased MDSC accumulation and increased CTL level. Our findings determine that 5-FU chemotherapy activates the p53-Fas pathway, to suppress MDSC accumulation, to increase CTL tumor infiltration.

Nrf3 alleviates oxidative stress and promotes the survival of colon cancer cells by activating AKT/BCL-2 signal pathway.

Oxidative stress is closely linked to tumor initiation and development, conferring a survival advantage to cancer cells. Therefore, understanding cancer cells' antioxidant molecular mechanisms is crucial to cancer therapy. In this study, we discovered that H2O2-induced oxidative stress increased Nrf3 expression in colon cancer cells. Overexpression of Nrf3 decreased H2O2-mediated cytotoxicity and apoptosis. Furthermore, Nrf3 reduced reactive oxygen species levels and malondialdehyde concentrations after H2O2 treatment. Mechanistically, H2O2-mediated cell apoptosis involves multiple signaling proteins, including Akt, bcl-2, JNK, and p38. An increase in Nrf3 expression in colon cancer cells treated with H2O2 partly reversed Akt/Bcl-2 inhibition, whereas it decreased activation of p38 and JNK. In addition, we found that increasing Nrf3 decreased stress-associated chemical-induced cell death, resulting in drug resistance. According to these results, Nrf3 is critical for drug resistance and oxidant adaptation.

Isoproterenol Alters Metabolism, Promotes Survival and Migration in 5-Fluorouracil-Treated SW480 Cells with and without Beta-hydroxybutyrate.

People with cancer often experience long-term physical and psychological stress, which can have a significant impact on tumor metabolism and treatment. The effects of adrenergic signaling on metabolic pathways are well known, but only a few studies have looked into the connection between this signaling and tumor metabolism. This study examined the effects of treatment with isoproterenol (Iso) alone and in combination with β-hydroxybutyrate (βHB), a mitochondrial fuel, on the metabolism, survival, and migration of SW480 colon cancer cells treated with 5-fluorouracil (5FU). The researchers measured the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) to determine the metabolic profile of these cells. They also analyzed the gene expression of PGC-1α, c-MYC, and NANOG to investigate the relationship between metabolic phenotype and stemness status. Scratch assays were used to assess cell migration. The results showed that Iso treatment increased cell viability in both SW480 and 5FU-treated SW480 cells. There was a significant decrease in ECAR and an increase in OCR after Iso treatment in both cell types. The expression of c-MYC and NANOG, genes associated with stemness, increased, while the expression of PGC-1α, a gene related to oxidative phosphorylation, decreased following Iso treatment. Iso treatment also increased the migration potential of both SW480 and 5FU-treated SW480 cells. These findings suggest that under stressful conditions, 5FU-treated colon cancer cells can utilize the oxidative phosphorylation pathway for growth and migration.

Dominant-Negative Form of SIGIRR: SIGIRRΔE8 Promotes Tumor Growth Through Regulation of Metabolic Pathways

Colorectal carcinoma is the leading cause of cancer-related death. Previously we have shown that tumor suppressor single immunoglobulin interleukin-1-related receptor (SIGIRR) is frequently inactivated in human colorectal cancer by the increased expression of a novel SIGIRR isoform (SIGIRRΔE8). SIGIRRΔE8 showed increased retention in the cytoplasm and loss of complex glycan modification compared to the full-length SIGIRR. Now we found that the arginine residues located in the C-terminus of SIGIRRΔE8 serve as an endoplasmic reticulum retention signal and are required for resident protein ribophorin 1 (RPN1) interaction. In addition, we found that SIGIRRΔE8 exerts a direct impact on cell metabolism through interaction with the adenosine triphosphate synthase in the colorectal cancer cells. SIGIRRΔE8 expression promoted the metabolic shift through upregulation of mammalian target of rapamycin signaling pathway and dysregulation of mitochondrial function to promote survival and proliferation of colon cancer cells in xenograft model.

AKT inhibition sensitizes EVI1 expressing colon cancer cells to irinotecan therapy by regulating the Akt/mTOR axis.

Ecotropic viral integration site 1 (EVI1) is an oncogenic transcription factor that has been attributed to chemotherapy resistance in different cancers. As yet, however, its role in colon cancer drug resistance is not completely understood. Here, we set out to investigate the functional and therapeutic relevance of EVI1 in colon cancer drug resistance. The EVI1 gene was knocked down in colon cancer cells that were subsequently tested for susceptibility to irinotecan using in vitro assays and in vivo subcutaneous mouse colon cancer models. The effect of EVI1 knockdown on the AKT-mTOR signaling pathway was assessed using cell line models, immunohistochemistry and bioinformatics tools. The anti-proliferative activity of AKT inhibitor GSK690693 and its combination with irinotecan was tested in colon cancer cell line models (2D and 3D). Finally, the therapeutic efficacy of GSK690693 and its combination with irinotecan was evaluated in xenografted EVI1 expressing colon cancer mouse models. We found that EVI1 knockdown decreased cancer stem cell-like properties and improved irinotecan responses in both cell line and subcutaneous mouse models. In addition, we found that EVI1 downregulation resulted in inhibition of AKT/mTOR signaling and RICTOR expression. Knocking down RICTOR expression increased the cytotoxic effects of irinotecan in EVI1 downregulated colon cancer cells. Co-treatment with irinotecan and ATP-competitive AKT inhibitor GSK690693 significantly reduced colon cancer cell survival and tumor progression rates. Inhibition of the AKT signaling cascade by GSK690693 may serve as an alternative to improve the irinotecan response in EVI1-expressing colon cancer cells.

Abstract 2552: Effects of liver endothelium on pancreatic cancer growth and metabolism

Abstract Introduction: ~90% metastatic pancreatic ductal adenocarcinoma (mPDAC) are found in the liver, and 5-year survival rate for patients with mPDAC is only at 3%. Therefore, novel therapeutic strategies are urgently needed. A growing body of evidence suggest PDAC rely on mitochondrial function (oxidative phosphorylation, OXPHOS) for survival. However, PDAC liver metastases have been reported to have higher levels of glucose uptake compared to primary tumors suggesting that PDAC liver metastases may rely on glycolytic metabolism. Therefore, the relative metabolic and cellular profiles of primary and metastatic PDAC (mPDAC) remain unclear and the involved regulatory pathway(s) have not been elucidated. Our previous studies showed that liver endothelial cells (ECs) secreted soluble factors to promote the survival of colon cancer cells in a paracrine fashion. The influence of the liver EC microenvironment on mPDAC growth and metabolism has not been elucidated. In this study, we investigate the paracrine effects of liver ECs on the survival and metabolic profiles of PDAC and elucidate the involved mechanism(s). Methods: Primary liver ECs were isolated from non-neoplastic liver. Conditioned medium (CM) from liver ECs were collected and then applied to PDAC cells, with CM from PDAC as control CM. Effects of CM on PDAC cell proliferation were measured by the MTT assay. Changes in phosphorylation of receptor tyrosine kinases (RTK) between PDAC CM and EC CM treated PADC cells were determined by a Phospho-RTK Array kit and then validated by Western blotting. Effects of EC CM on PDAC metabolism was assessed by ATP production with CellTiter-Glo and oxygen consumption rate with Agilent Seahorse Mito Stress Assay. Results: Compared to PDAC CM, liver EC CM promoted proliferation in different PDAC cells. We found that human epidermal growth factor receptor 3 (HER3, also known asERBB3) was only expressed and activated in BxPC-3 cells (HER3+ve), in which the HER3-AKT signaling pathway was activated by EC CM. Furthermore, blocking HER3 activation with a humanized HER3 antibody, seribantumab, significantly blocked EC CM-induced AKT activation and cell proliferation. Moreover, depletion of neuregulin (NRG) from EC CM attenuated HER3-AKT activation and indicated that EC-secreted NRG might play a role in promoting PDAC growth. Furthermore, EC CM decreased the levels of ATP production and O2 consumption in PDAC cells, suggesting that EC cells reprogram PDAC metabolism away from OXPHOS mitochondrial metabolism. Conclusions: Liver EC-secreted factors promoted PDAC growth in vitro and in vivo, and HER3 was expressed in a subset of PDAC cells and mediated EC-induced proliferation. Moreover, EC reprogramed PDAC metabolism towards glycolysis, and inhibiting the activation of HER3 shifted PDAC metabolism to OXPHOS. Our findings provide a new insight to develop new combination of HER antibody and OXPHOS inhibitor for treating patients with HER3+ve mPDAC. Citation Format: Wei Zhang, Michel'le Wright, Moeez Rathore, Mehrdad Zarei, Jordan Winter, Rui Wang. Effects of liver endothelium on pancreatic cancer growth and metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2552.

Abstract 864: Ophiopogonin D increase apoptosis by activating p53 via ribosomal protein L5 and L11 and inhibiting the expression of c-Myc via CNOT2

Abstract Extracted from the root tuber of Ophiopogon japonicas, ophiopogon is well known to have an anti-cancer effect. However, the underlying mechanisms are still largely unknown. Here, we report that Ophiopogon D (OP-D) can inhibit colon cancer cell proliferation and induce apoptosis by inhibiting c-Myc expression through activation of p53 and CNOT2 regulation. Our results showed that OP-D induced p53 expression via ribosomal protein L5 or L11 and inhibited c-Myc expression through CNOT2 in a dose-dependent manner. Additionally, OP-D regulated cyclin D1 and CDK4 which are well known as cell cycle regulatory proteins. Consistently, OP-D inhibited the phosphorylation of AKT expression in a dose-dependent manner. Furthermore, OP-D shortened c-Myc’s half-life in a time-dependent manner. Furthermore, CNOT2 knockdown enhanced the inhibitory effect of OP-D on c-Myc in colon cancer cells. Interestingly, OP-D has increased the apoptotic effect of colon cancer cells when combined with doxorubicin or 5-FU, a treatment already used clinically. Altogether, our results suggested that OP-D regulates colon cancer cell survival and induces apoptosis by inhibiting c-Myc expression via activation of p53 and CNOT2 regulation. Citation Format: Hyun Min Ko, Wona Jee, Do-il Park, Somi Park, Ye-Rin Park, Hyeung-Jin Jang, Ji Hoon Jung. Ophiopogonin D increase apoptosis by activating p53 via ribosomal protein L5 and L11 and inhibiting the expression of c-Myc via CNOT2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 864.

Deoxyelephantopin Induces Apoptosis and Enhances Chemosensitivity of Colon Cancer via miR-205/Bcl2 Axis.

Colon cancer (CC) is one of the major causes of cancer death in humans. Despite recent advances in the management of CC, the prognosis is still poor and a new strategy for effective therapy is imperative. Deoxyelephantopin (DET), extracted from an important medicinal plant, Elephantopus scaber L., has been reported to exhibit excellent anti-inflammatory and -cancer activities, while the detailed anti-cancer mechanism remains unclear. Herein, we found that DET showed a significant CC inhibiting effect in vitro and in vivo without obvious organ toxicity. Mechanistically, DET inhibited CC cells and tumor growth by inducing G2/M phase arrest and subsequent apoptosis. DET-mediated cell cycle arrest was caused by severe DNA damage, and DET decreased the Bcl2 expression level in a dose-dependent manner to promote CC cell apoptosis, whereas restoring Bcl2 expression reduced apoptosis to a certain extent. Moreover, we identified a microRNA complementary to the 3′-UTR of Bcl2, miR-205, that responded to the DET treatment. An inhibitor of miR-205 could recover Bcl2 expression and promoted the survival of CC cells upon DET treatment. To further examine the potential value of the drug, we evaluated the combinative effects of DET and 5-Fluorouracil (5FU) through Jin’s formula and revealed that DET acted synergistically with 5FU, resulting in enhancing the chemotherapeutic sensitivity of CC to 5FU. Our results consolidate DET as a potent drug for the treatment of CC when it is used alone or combined with 5FU, and elucidate the importance of the miR-205-Bcl2 axis in DET treatment.

Role of endoplasmic reticulum stress in apoptosis induced by HK2 inhibitor and its potential as a new drug combination strategy.

Compared with normal cells, tumor cells mainly obtain energy through aerobic glycolysis. Hexokinase 2 (HK2) plays a key role in the regulation of tumor cell aerobic glycolysis, and targeting HK2 has become a new strategy for cancer treatment. However, little is known about the role of HK2 in colon cancer and the regulation of its targeted inhibitors. In this study, we found that the expression of HK2 in colorectal cancer tissues was significantly higher than that in adjacent tissues, and the expression level of HK2 in metastatic colorectal cancer was further increased. Meanwhile, the expression level of HK2 was closely related to clinical TNM stage and outcome of colorectal cancer patients. We provide here evidence that HK2 inhibitor 3-Bromopyruvate acid (3-BP) can significantly inhibit the survival and proliferation of colon cancer cells, and induce apoptosis through mitochondrial apoptosis signaling pathway. In addition, we found that 3-BP can also induce endoplasmic reticulum stress in colon cancer cells, the mechanism may be through the increase of intracellular calcium concentration. In vitro and in vivo experiments showed that inhibition of endoplasmic reticulum stress could further increase the proliferation inhibition and apoptosis induced by 3-BP. Collectively, our results show that HK2 is highly expressed in colorectal cancer. 3-BP, an inhibitor of HK2, can induce apoptosis and endoplasmic reticulum stress in colon cancer cells. Endoplasmic reticulum stress plays a protective role in cell death induced by 3-BP. This result suggested that targeting HK2 and endoplasmic reticulum stress may be a valuable strategy in targeted and combination therapy of colon cancer.

Apoptotic Effect of Brassinin via Inhibition of CNOT2 and Activation of p53 and Its Combination Effect with Doxorubicin

Brassinin derived from Chinese cabbage has been reported to act as an anti-cancer agent on prostate, liver, and colon cancer cells. However, its mechanism and impact are largely unknown in colon cancer cells. Here, we first published a report that Brassinin induces apoptosis and inhibits the survival of colon cancer cells by activating p53. We found that Brassinin induces p53 and p21 dose- and time-dependent manner in wild type of p53 colon cancer cells. Interestingly, Brassinin induces apoptosis in wild-type of p53 cancer cells, but not in null-type of p53 cancer cells dose dependently. Additionally, Brassinin induces apoptosis through L5. Furthermore, Brassinin enhanced the apoptotic effect with doxorubicin by activating p53. Altogether, our findings suggest that Brassinin is a new p53 regulator via induce apoptosis and inhibit the proliferation in colon cancer cells.

Role of UPR Sensor Activation in Cell Death-Survival Decision of Colon Cancer Cells Stressed by DPE Treatment.

Polyphenols have been shown to possess several beneficial properties, including properties involved in the prevention or treatment of cancer. Among these polyphenols, a leading role is played by dihydroxyphenylethanol (DPE), the most powerful antioxidant compound contained in the olive oil. DPE has been previously reported to induce endoplasmic reticulum (ER) stress and to reduce cell survival in colon cancer, one of the most common and aggressive cancers in developed countries. In this study, we further investigated the activation of UPR by DPE and explored the roles of the three UPR sensors, inositol-requiring enzyme (IRE) 1 alpha, protein kinase RNA-like endoplasmic reticulum kinase (PERK), and activating transcription factor (ATF6), in the cell death–survival decision of wt and mutp53 colon cancer cells and the underlying mechanisms involved. We also unveiled a new interplay between ATF6 and wt, as well as mutp53, which may have important implications in cancer therapy.

PD-1-siRNA Delivered by Attenuated Salmonella Enhances the Antitumor Effect of Chloroquine in Colon Cancer.

The widespread appearance of drug tolerance and the low efficiency of single treatment have severely affected the survival time of the patients with colorectal cancer. Exploring new treatment options and combined treatment strategies have become the key to improving the prognosis. The combination of immunotherapy and chemotherapy have shown good clinical expectations. Here, we studied the cooperative effects of chloroquine, an anti-malarial drug that is now widely used in anti-tumor research, and RNA interference (RNAi) targeting the immune checkpoint molecule Programmed Death-1 (PD-1) delivered with attenuated Salmonella. Our results show that chloroquine can not only significantly inhibit the survival of colon cancer cells and induce apoptosis, but also effectively inhibit cell invasion and migration. The results of in vivo experiments show that chloroquine can increase the expression of PD-1 in tumor tissues. Combining chloroquine and PD-1 siRNA can further inhibit the growth and metastases of colon cancer and induce apoptosis. The mechanism underlying this phenomenon is the occurrence of chloroquine-induced apoptosis and the effective immune response caused by the attenuated Salmonella carrying PD-1 siRNA. This study suggests that the combined application of PD-1-based immunotherapy and anti-cancer drugs has become a new expectation for clinical treatment of colorectal cancer.

Abstract 936: Albendazole enhances apoptosis through targeting RNF20/Eg5 interaction in colon cancer cells

Abstract Colorectal cancer (CRC) is one of the most leading causes of cancer-related death in the world because most of cases are diagnosed in advanced stages, when the development of resistance to chemotherapy is more frequent. To face this situation, new therapeutic drugs are desperately needed for patients with advanced cancer, but making safe and efficacious drugs remains expensive and time consuming. Given the high attrition rates, substantial costs and slow pace of new drug discovery and further development, repurposing of 'old' drugs is increasingly becoming an attractive proposition. The objective of this study was to perform a high-throughput screen (HTS) of an FDA-approved drug library (1622 compounds) to specifically identify candidates that can significantly reduce the cell survival of colon cancer cells. Among all, Albendazole which is an anthelmintic drug demonstrated promising in vitro efficacy against colon cancer cells with relatively low toxicity in normal cells. Albendazole is known to bind to the colchicine-sensitive site of β-tubulin, inhibiting their polymerization into microtubules. The decrease in microtubules in the intestinal cells of the parasites decreases their absorptive function, especially the uptake of glucose by the adult and larval forms of the parasites, and also depletes glycogen storage. Insufficient glucose results in insufficient energy for the production of adenosine triphosphate (ATP) and thus parasite dies eventually during helminthic infection. Here, we determined to molecular mechanism/s by which Albendazole regulates colon cancer cell viability. To explore the global changes in colon cancer cells, we performed mass spectrometry analysis in colon cancer cells treated with Albendazole. One of the major proteins which was downregulated by Albendazole was RNF20. RNF20 is a E3 ubiquitin ligase catalyzing histone H2B monoubiquitination, interacts with the motor protein Eg5 during mitosis and participates in spindle assembly. Inhibition of these proteins induces apoptosis by interfering with antiapoptotic proteins Bcl2 family and arrests cells remarkably at G2/M phase in colon cancer cells. Furthermore, Albendazole inhibits invasion and colony formation in vitro, induced apoptosis in tumoroids derived from APCmin mice (Spontaneous colon cancer model) and xenograft tumor formation in vivo. These results indicate that albendazole may have therapeutic potential in colon cancer and provide the framework for future studies of albendazole in CRC. Citation Format: Iram Fatima, Rizwan Ahmad, Saiprasad Gowrikumar, Susmita Barman, Rafay Abu, Vikas Kumar, Amar B. Singh, Punita Dhawan. Albendazole enhances apoptosis through targeting RNF20/Eg5 interaction in colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 936.