Colorectal Cancer Cell Death Research Articles

Anticancer Activity of Camel Milk via Induction of Autophagic Death in Human Colorectal and Breast Cancer Cells

Background/Objective:Camel milk is traditionally known for its human health benefits and believed to be a remedy for various human ailments including cancer. The study was aimed to evaluate the inhibitory effects of commercially available camel milk on cancer cells and its underlying mechanism(s).Materials and Methods:Two cell lines: colorectal cancer HCT 116 and breast cancer MCF-7 were cultured with different doses of camel milk. The effects of camel milk on cell death were determined by MTT assay, viability by trypan blue exclusion assay and migration by in vitro scratch assay. The mechanism was elucidated by western blotting and confocal microscopy was used to confirm autophagy.Results:Camel milk significantly reduced proliferation, viability as well as migration of both the cells. The accumulation of LC3-II protein along with reduction in expression of p62 and Atg 5-12, the autophagy proteins implied induction of autophagy. The (GFP)-LC3 puncta detected by confocal microscopy confirmed the autophagosome formation in response to camel milk treatment.Conclusion:Camel milk exerted antiproliferative effects on human colorectal HCT 116 and breast MCF-7 cancer cells by inducing autophagy.

RSL3 Drives Ferroptosis Through GPX4 Inactivation and ROS Production in Colorectal Cancer.

Ferroptosis is an iron-dependent, oxidative cell death, and is characterized by iron-dependent accumulation of reactive oxygen species (ROS) within the cell. It has been implicated in various human diseases, including cancer. Recently, ferroptosis, as a non-apoptotic form of cell death, is emerging in specific cancer types; however, its relevance in colorectal cancer (CRC) is unexplored and remains unclear. Here, we showed that ferroptosis inducer RSL3 initiated cell death and ROS accumulation in HCT116, LoVo, and HT29 CRC cells over a 24 h time course. Furthermore, we found that ROS levels and transferrin expression were elevated in CRC cells treated with RSL3 accompanied by a decrease in the expression of glutathione peroxidase 4 (GPX4), indicating an iron-dependent cell death, ferroptosis. Overexpression GPX4 resulted in decreased cell death after RSL3 treatment. Therefore, RSL3 was able to induce ferroptosis on three different CRC cell lines in vitro in a dose- and time-dependent manner, which was due to increased ROS and an increase in the cellular labile iron pool. Moreover, this effect was able to be reversed by overexpression of GPX4. Taken together, our results suggest that the induction of ferroptosis contributed to RSL3-induced cell death in CRC cells and ferroptosis may be a pervasive and dynamic form of cell death for cancer treatment.

TRIB3 Interacts With β-Catenin and TCF4 to Increase Stem Cell Features of Colorectal Cancer Stem Cells and Tumorigenesis

Activation of Wnt signaling to β-catenin contributes to the development of colorectal cancer (CRC). Expression of tribbles pseudo-kinase 3 (TRIB3) is increased in some colorectal tumors and associated with poor outcome. We investigated whether increased TRIB3 expression promotes stem cell features of CRC cells and tumor progression by interacting with the Wnt signaling pathway. We performed studies with C57BL/6J-ApcMin/J mice injected with an adeno-associated virus vector that expresses a small hairpin RNA against Trib3 mRNA (ApcMin/J-Trib3KD) or a control vector (ApcMin/J-Ctrl). We created BALB/c mice that overexpress TRIB3 from an adeno-associated virus vector and mice with small hairpin RNA-mediated knockdown of β-catenin. The mice were given azoxymethane followed by dextran sodium sulfate to induce colitis-associated cancer. Intestinal tissues were collected and analyzed by histology, gene expression profiling, immunohistochemistry, and immunofluorescence. Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5)-positive (LGR5Pos) and LGR5-negative (LGR5Neg) HCT-8 CRC cells, with or without knockdown or transgenic expression of TRIB3, were sorted and analyzed in sphere-formation assays. We derived organoids from human and mouse colorectal tumors to analyze the function of TRIB3 and test the effect of a peptide inhibitor. Wnt signaling to β-catenin was analyzed in dual luciferase reporter, chromatin precipitation, immunofluorescence, and immunoblot assays. Proteins that interact with TRIB3 were identified by immunoprecipitation. CRC cell lines were grown in nude mice as xenograft tumors. At 10 weeks of age, more than half the ApcMin/J-Ctrl mice developed intestinal high-grade epithelial neoplasia, whereas ApcMin/J-Trib3KD mice had no intestinal polyps and normal histology. Colon tissues from ApcMin/J-Trib3KD mice expressed lower levels of genes regulated by β-catenin and genes associated with cancer stem cells. Mice with overexpression of Trib3 developed more tumors after administration of azoxymethane and dextran sodium sulfate than BALB/c mice. Mice with knockdown of β-catenin had a lower tumor burden after administration of azoxymethane and dextran sodium sulfate, regardless of Trib3 overexpression. Intestinal tissues from mice with overexpression of Trib3 and knockdown of β-catenin did not have activation of Wnt signaling or expression of genes regulated by β-catenin. LGR5Pos cells sorted from HCT-8 cells expressed higher levels of TRIB3 than LGR5Neg cells. CRC cells that overexpressed TRIB3 had higher levels of transcription by β-catenin and formed larger spheroids than control CRC cells; knockdown of β-catenin prevented the larger organoid size caused by TRIB3 overexpression. TRIB3 interacted physically with β-catenin and transcription factor 4 (TCF4). TRIB3 overexpression increased, and TRIB3 knockdown decreased, recruitment of TCF4 and β-catenin to the promoter region of genes regulated by Wnt. Activated β-catenin increased expression of TRIB3, indicating a positive-feedback loop. A peptide (P2-T3A6) that bound β-catenin disrupted its interaction with TRIB3 and TCF4. In primary CRC cells and HCT-8 cells, P2-T3A6 decreased expression of genes regulated by β-catenin and genes associated with cancer stem cells and decreased cell viability and migration. Injection of C57BL/6J-ApcMin/J mice with P2-T3A6 decreased the number and size of tumor nodules and colon expression of genes regulated by β-catenin. P2-T3A6 increased 5-fluorouracil-induced death of CRC cells and survival times of mice with xenograft tumors. TRIB3 interacts with β-catenin and TCF4 in intestine cells to increase expression of genes associated with cancer stem cells. Knockdown of TRIB3 decreases colon neoplasia in mice, migration of CRC cells, and their growth as xenograft tumors in mice. Strategies to block TRIB3 activity might be developed for treatment of CRC.

Polydopamine-coated Au-Ag nanoparticle-guided photothermal colorectal cancer therapy through multiple cell death pathways

Nanoparticles are emerging as a new therapeutic modality due to their high stability, precise targeting, and high biocompatibility. Branched Au-Ag nanoparticles with polydopamine coating (Au-Ag@PDA) have strong near-infrared absorbance and no cytotoxicity but high photothermal conversion efficiency. However, the photothermal activity of Au-Ag@PDA in vivo and in vitro has not been reported yet, and the mechanism underlying the effects of photothermal nanomaterials is not clear. Therefore, in this study, the colorectal cancer cell line HCT-116 and nude mice xenografts were used to observe the photothermal effects of Au-Ag@PDA in vivo and in vitro. The results suggest that Au-Ag@PDA NPs significantly inhibited cell proliferation and induced apoptosis in colorectal cancer cells. Moreover, Au-Ag@PDA NP-mediated photothermal therapy inhibited the growth of tumors at doses of 50 and 100 μg in vivo. The mechanisms through which Au-Ag@PDA NPs induced colorectal cancer cell death involved multiple pathways, including caspase-dependent and -independent apoptosis, mitochondrial damage, lysosomal membrane permeability, and autophagy. Thus, our findings suggest that Au-Ag@PDA NPs could be used as potential antitumor agents for photothermal ablation of colorectal cancer cells.

Tanshinone IIA promotes IL2-mediated SW480 colorectal cancer cell apoptosis by triggering INF2-related mitochondrial fission and activating the Mst1-Hippo pathway.

IL-2-based therapy is a promising tool to treat colorectal cancer, but drug resistance always occurs in clinical practice. Mitochondrial fission is a novel target to modulate cancer development and progression. The aim of our study is to explore the effect of IL-2 combined with Tan IIA on SW480 colorectal cancer cell apoptosis in vitro and to determine whether IL-2/Tan IIA cotreatment could reduce SW480 cell viability via activating mitochondrial fission. The results indicated that Tan IIA increased IL-2-mediated cell death in SW480 colorectal cancer cells, and this effect was also accompanied with a reduction in cell proliferation. Functional investigations demonstrated that Tan IIA/IL-2 cotreatment enhanced INF2-related mitochondrial fission. Excessive mitochondrial division induced mitochondrial oxidative stress, mitochondrial energy metabolism disorder and mitochondrial apoptosis in SW480 cells. Inhibition of mitochondrial fission attenuated the antitumor effect of Tan IIA/IL-2 cotreatment on SW480 cell apoptosis. Further, we demonstrated that Tan IIA/IL-2 combination therapy controlled INF2-related mitochondrial fission via the Mst1-Hippo pathway. Moreover, Mst1 knockdown abrogated Tan IIA/IL-2-activated mitochondrial fission. Altogether, our results demonstrated that Tan IIA enhances the therapeutic efficiency of IL-2-mediated SW480 colorectal cancer cell apoptosis via promoting INF2-related mitochondrial fission and activating the Mst1-Hippo pathway.

558P - The combination of trifluridine/tipiracil and oxaliplatin induces immunogenic cell death in microsatellite stable colorectal cancer

558P - The combination of trifluridine/tipiracil and oxaliplatin induces immunogenic cell death in microsatellite stable colorectal cancer

Discovery of a small molecule targeting autophagy via ATG4B inhibition and cell death of colorectal cancer cells in vitro and in vivo

ABSTRACTHuman Atg4 homologs are cysteine proteases, which play key roles in the macroautophagy/autophagy process by cleaving Atg8 homologs for conjugation to lipid membranes and for deconjugation of Atg8 homologs from membranes. Expression of ATG4B is significantly increased in colorectal cancer cells compared to normal cells, suggesting that ATG4B may be important for cancer biology. Inhibition of ATG4B may reduce the autophagy activity, thereby sensitizing cancer cells to therapeutic agents. Thus, developing specific and potent ATG4B inhibitors for research as well as for potential therapeutic uses is highly needed. In this study, we integrated in silico screening and in vitro assays to discover a potent ATG4B inhibitor, named S130, from a noncommercial library. This chemical binds to ATG4B with strong affinity and specifically suppresses the activity of ATG4B but not other proteases. S130 did not cause the impairment of autophagosome fusion, nor did it result in the dysfunction of lysosomes. Instead, S130 might attenuate the delipidation of LC3-II on the autolysosomes to suppress the recycling of LC3-I, which normally occurs after LC3-II cleavage by ATG4B. Intriguingly, S130 induced cell death, which was accompanied with autophagy stress and could be further exacerbated by nutrient deprivation. Such cytotoxicity could be partially reversed by enhancing ATG4B activity. Finally, we found that S130 was distributed in tumor tissues in vivo and was also effective in arresting the growth of colorectal cancer cells. Thus, this study indicates that ATG4B is a potential anticancer target and S130 might be a novel small-molecule candidate for future cancer therapy.

Blue light emitting diodes irradiation causes cell death in colorectal cancer by inducing ROS production and DNA damage.

The light emitting diodes (LEDs) irradiation has been demonstrated to be potential therapeutic strategies for several diseases. However, the blue LED effects remain largely unknown in colorectal cancer (CRC), which is a major cause of morbidity and mortality throughout the world. In this study, we determined the effects of blue LED irradiation, the maximal light emission at 470 nm in wavelength, in human CRC cell lines SW620 and HT29. The cells were irradiated with blue LED light for 0 J/cm2, 72 J/cm2, 144 J/cm2, 216 J/cm2 and 288 J/cm2 respectively. We found that irradiation with blue LED light induced a marked decrease of live cells and an increase of dead cells. Additionally, lower cell proliferation and a remarkably increase of cell apoptosis were observed in blue LED-irradiated cells as compared with non-irradiated control group. The cell migration was significantly inhibited by blue LED irradiation 24, 48 and 72 h later compared with non-treated group. Blue LED-treated CRC cells further displayed a remarkably inhibition of EMT process in CRC cells. Finally, we found the accumulation of ROS production and DNA damage were induced by blue LED irradiation. These results indicated that blue LED irradiation inhibits CRC cell proliferation, migration and EMT process as well as induces cell apoptosis, which may result from increased ROS accumulation and induction of DNA damage.

Abstract LB-080: Discovery of an unconventional form of cell-death in colorectal cancer

Abstract We recently discovered unique, structurally constrained 4-pyridinyl - containing compounds that induce a form of caspase-independent cell death in colorectal cancer (CRC) cells. This was characterized by the simultaneous induction of dysregulated macropinocytosis and macroautophagy, a type of cell death we have termed ‘methuophagy' (this neologism arises from the Greek ‘methuo', to drink to intoxication and ‘phagy', self-eating). Our new class of small molecular probes (BAPT analogues) do not induce cell death via activation of the well characterized classical apoptotic pathways. In contrast, our BAPT compounds cause methuophagy by inducing the formation and accumulation of fluid-filled, heterogeneous vacuoles and autophagosomes, resulting in metabolic failure, loss of membrane integrity, and detachment of cells from the substratum. Methuophagy produces extensive autophagy without cell shrinkage, chromatin condensation, alteration of mitochondrial membrane potential or nuclear membrane damage. The optimization of the BAPT structure led to the discovery of BAPT-27, which has nanomolar potency in inducing methuophagy in broad spectrum of CRC cell lines and may serve as a prototype for a new class of drugs. BAPT-27 produces methuophagy and surmounts MDR in CRC cells independent of Ras, p53 or other apoptotic protein (i.e. Bax, Bak) or expression of ATP-binding cassette (ABC) transporters. Furthermore, BAPT-27 is non-toxic in normal cells and zebrafish. Based on our findings, we postulate that the induction of methuophagy by BAPT-27 produces endolysosomal trafficking defects that prevent recycling of lysosomes and cause lysosome-to-nucleus signaling defect, a mechanism that disturb cellular clearance and energy metabolism. The selected compounds will allow us to understand the biology of methuophagy-induced cell death and will be particularly beneficial for treatment of patients with apoptotic-resistant and aggressive CRCs. Citation Format: Amit K. Tiwari, Haneen Amawi, Karthikeyan Chandrabose, Paul Erhardt, Piyush Trivedi. Discovery of an unconventional form of cell-death in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-080.

Abstract 2675: Quinacrine-mediated activation of JNK1 inhibits Nrf2 and causes cell death in human colon cancer

Abstract Up-regulation of Nrf2 is associated with poor prognosis in skin, breast, prostate, lung, head and neck, endometrial, and colorectal cancer, and increases chemoresistance and radioresistance by inducing antioxidant and detoxification genes. Nrf2 inhibitors are not currently available for clinical use. Here, we investigated the feasibility of using quinacrine, an old antimalarial drug, as a novel Nrf2 inhibitor for treating colorectal cancer. Immunohistochemical analysis of colorectal cancer samples from 60 patients was used to assess the correlation between Nrf2 expression levels and clinicopathological features. Using data from tumors in the Cancer Genome Atlas (n=290 normal, n=295 cancer) and publicly available data sets, we validated the correlation between Nrf2 expression and poor prognosis in colorectal cancer. The effect of quinacrine on the activation of JNK1-mediated Nrf2 degradation was analyzed with co-immunoprecipitation, immunoblotting, and luciferase reporter assays. To confirm that quinacrine-mediated Nrf2 degradation caused cancer cell death, we used Nrf2 gain-of-function and loss-of-function experiments. The cytotoxicity of quinacrine against colorectal cancer cells under both normoxia (20% O2) and hypoxia (0.5% O2) was assessed by clonogenic survival assays. Finally, a xenograft mouse model was used to confirm the results of our in vitro experiments in an in vivo setting. High Nrf2 levels were correlated with increased expression of its target genes and poor survival among colorectal cancer patients in immunohistochemical analysis and analysis for publicly available data sets. The quinacrine-mediated activation of JNK1 induced Nrf2 degradation by increasing the interaction between Keap1 and Nrf2, thereby causing cancer cell death under normoxia and hypoxia and suppressing tumor growth in our in vivo model. Overexpression of Nrf2 effectively prevented quinacrine-mediated Nrf2 degradation and cancer cell death regardless of oxygen tension. Additionally, siRNA-mediated JNK1 knockdown inhibited quinacrine-mediated Nrf2 degradation. Luciferase reporter assays and qPCR revealed that quinacrine inhibited Nrf2-mediated transcription in cancer cells under normoxia and hypoxia. Finally, the quinacrine-mediated degradation of Nrf2 was found to increase the sensitivity of colorectal cancer in in vivo model and cancer cells under both normoxia and hypoxia in vitro to ROS-inducing drugs and anticancer drugs commonly used against colorectal cancer. Quinacrine caused colorectal cancer cell death by promoting activation of JNK1-mediated Nrf2 degradation. Citation Format: Eun-Taex Oh, Chan-Woo Kim, Ha Gyeong Kim, In-Seon Lee, Chae Won Lee, Heon Joo Park. Quinacrine-mediated activation of JNK1 inhibits Nrf2 and causes cell death in human colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2675.

PO-114 Overexpression of miR-205–3 p increases sensitivity to low-dose ionising radiation in DLD-1 cells

IntroductionEnhanced radiosensitivity at low doses of ionising radiation (IR) (0.2 to 0.6 Gy) has been reported in several cell lines. This phenomenon, known as low doses hyper–radiosensitivity (LDHRS), appears as an opportunity to decrease toxicity of radiotherapy. However, this effect is subtle and the mechanism underlying LDHRS has not been clearly explained, limiting the benefit of LDHRS in clinical practice.Material and methodsTo determinate LDHRS in DLD–1 human colorectal cancer cells, viability, DNA damage, cell death and apoptosis were evaluated by: MTS, γ–H2AX immunofluorescence, trypan blue dye assays, Caspase 3/7 activity and hypoploidy determination; in cells exposed to low (0.6 Gy) and high (12 Gy) doses IR. LDHRS in DLD.-1 cells was confirmed by clonogenic assays and mathematical modelling. Then, to understand the mechanisms responsible for cell death induced by low-dose IR, we evaluated a panel of 86 miRNAs in array-PCR format. Validation of targets was made by qRT-PCR and functional assays were achieved by transient overexpression.Results and discussionsOur results show that DLD–1 cells display LDHRS. Also, doses of 0.6 Gy induced an early but transient DNA damage response. miRNA expression assays revealed an overexpression of 3 microRNAs (miR-205–3 p, miR–1 and miR–133b) and down regulation of 2 miRNAs (miR–122–5 p, and miR–134–5 p). This miRNA profile differed from the one in cells exposed to high-dose IR (12 Gy). Transfection with mimetic of miR-205–3 p, the most overexpressed miRNA in cells exposed to 0.6 Gy, induced cell death at lower dose, increasing LDHRS response. Interestingly, at higher doses (12 Gy), miR–205–3 p induced formation of multi- and micronuclei, features associated with mitotic catastrophe. Thus, IR evoked differents miRNAs profiles, in agreement with a distinct low dose radiation-induced cell death mechanism. Moreover, MiR–205–3 p increased radiosentitivity by two-ways: at low doses, by apoptosis induction, and higher doses by mitotic catastrophe. This effect could be used to boost the outcome of IR, while decreasing therapeutic doses.ConclusionWe propose miR-205–3 p as a potential radiosensitizer to low-dose IR.AcknowledgmentsThis work was supported by grant Anillo ACT115, PIA Program, CONICYT, FONDECYT 1151435, 1160889 and the national doctoral fellowship 21130246 CONICYT

Ellagitannins from Pomegranate Ameliorates 5-Fluorouracil-Induced Intestinal Mucositis in Rats while Enhancing Its Chemotoxicity against HT-29 Colorectal Cancer Cells through Intrinsic Apoptosis Induction

Worldwide, colorectal cancer (CRC) is a deleterious disease causing millions of death annually. 5-Fluorouracil (5-FU) is a first-line chemotherapy for CRC, but chemoresistance and gastrointestinal mucositis limit its efficacy. Polyphenol-rich foods are increasingly popular due to their potential beneficial roles in preventing and treating cancer. Ellagitannins are a group of phenolic compounds commonly found in pomegranate, strawberries, raspberries, etc. The objective of this study was to explore whether ellagitannins from pomegranate (PETs) could ameliorate 5-FU-induced intestinal mucositis and enhance the drug's efficacy against CRC. The results showed that PETs (100 mg/kg) counteracted 5-FU-induced intestinal mucositis in rats. The number of apoptotic cells per crypt was reduced from 1.50 ± 0.21 to 0.85 ± 0.18 ( P < 0.05). Moreover, PETs induced HT-29 CRC cell death through intrinsic apoptosis, as demonstrated by dissipation of mitochondrial membrane potential, increased Bax-to-Bcl-2 ratio, and cleavage of caspase 9 and caspase 3. PETs and 5-FU combination treatments exhibited synergistic cytotoxicity against HT-29 cells with a weighted combination index of 0.3494. PETs (80 μg/mL) and 5-FU (40 μg/mL) treatments for 48 h induced 14.03 ± 0.76% and 16.42 ± 1.15% of HT-29 cells to undergo apoptosis, while the combination treatment further increased apoptosis of cells to 34.00 ± 1.54% ( P < 0.05). Combination treatment of the cells also enhanced S phase cell cycle arrest as compared with PETs or 5-FU monotherapy ( P < 0.05). These results suggest that dietary ellagitannins from pomegranate could alleviate intestinal mucositis in rats induced by 5-FU while enhancing its toxicity against HT-29 cells through potentiation of apoptosis and cell cycle arrest.

Survivin antagonizes chemotherapy-induced cell death of colorectal cancer cells.

Irinotecan (CPT-11) and oxaliplatin (L-OHP) are among the most frequently used drugs against colorectal tumors. Therefore, it is important to define the molecular mechanisms that these agents modulate in colon cancer cells. Here we demonstrate that CPT-11 stalls such cells in the G2/M phase of the cell cycle, induces an accumulation of the tumor suppressor p53, the replicative stress/DNA damage marker γH2AX, phosphorylation of the checkpoint kinases ATM and ATR, and an ATR-dependent accumulation of the pro-survival molecule survivin. L-OHP reduces the number of cells in S-phase, stalls cell cycle progression, transiently triggers an accumulation of low levels of γH2AX and phosphorylated checkpoint kinases, and L-OHP suppresses survivin expression at the mRNA and protein levels. Compared to CPT-11, L-OHP is a stronger inducer of caspases and p53-dependent apoptosis. Overexpression and RNAi against survivin reveal that this factor critically antagonizes caspase-dependent apoptosis in cells treated with CPT-11 and L-OHP. We additionally show that L-OHP suppresses survivin through p53 and its downstream target p21, which stalls cell cycle progression as a cyclin-dependent kinase inhibitor (CDKi). These data shed new light on the regulation of survivin by two clinically significant drugs and its biological and predictive relevance in drug-exposed cancer cells.

Tanshinone IIA regulates colorectal cancer apoptosis via attenuation of Parkin‑mediated mitophagy by suppressing AMPK/Skp2 pathways.

Mitophagy is important for cancer development. Notably, the role of Parkin‑mediated mitophagy in colorectal cancer (CRC) mortality has not been fully determined. Therefore, the present study aimed to investigate the effect of Parkin‑mediated mitophagy on CRC apoptosis. In addition, the present study investigated the therapeutic effects of TanshinoneIIA (TanIIA) on the regulation of CRC cell death via mitophagy. Cellular apoptosis was measured following TanIIA treatment. In addition, mitophagy activity was evaluated by immunofluorescence and western blotting. The results of the present study revealed that TanIIA may enhance CRC cell death. In addition, the results demonstrated that TanIIA enhanced mitochondrial apoptosis, as demonstrated by reduced mitochondrial membrane potential, elevated mitochondrial permeability transition pore opening, and increased oxidative stress, mitochondrial energy disorder and proapoptotic factor expression. Furthermore, the results of the present study demonstrated that TanIIA induced mitochondrial apoptosis via inhibition of mitophagy. In addition, it was revealed that mitophagy could suppress mitochondrial apoptosis. Functional assays revealed that TanIIA suppressed the adenosine monophosphate‑activated protein kinase (AMPK) pathway, resulting in the inactivation of S‑phase kinase associated protein2 (Skp2). Furthermore, reduced levels of Skp2 failed to activate Parkin, thus resulting in inhibition of mitophagy. Conversely, reactivation of AMPK and overexpression of Skp2 rescued mitophagy activity and thus attenuated the TanIIA‑induced apoptosis of CRC cells. In conclusion, the results of the present study demonstrated the beneficial role of mitophagy in CRC cell survival and suggested that TanIIA may be an effective therapeutic agent, which suppresses mitophagy activity and enhances CRC apoptosis.

Screening and Identification of Compounds in Pomegranate Peel to Ameliorate 5‐Fluorouracil Induced Intestinal Mucositis in Rats and Induce HT‐29 Colorectal Cancer Cell Death through Reactive Oxygen Species Mediated Apoptosis and Cell Cycle Arrest

Upon searching the Chinese Pharmacopoeia, it is found that 97 herbs belong to the large intestine meridians among which 23 of them are traditionally used for intestine‐related cancer treatment. Pomegranate peel, in particular, is used to treat diarrhea, dysentery, and bloody stool which are similar to symptoms of colorectal cancer and intestinal mucositis. As such, pomegranate peel is selected for network pharmacology analysis. Pathways related to colorectal cancer, VEGF, toll‐like receptor and mTOR, together with gene‐associated diseases like cancer, immune disorders and infection were found to be significantly related to compounds in pomegranate peel. DPPH and ABTS free‐radical scavenging activity assays coupled with HPLC‐DAD‐MS/MS analysis were employed to identify the pomegranate peel extracts and corresponding compounds with the strongest antioxidant activities. Various concentrations of acetone, methanol, and ethanol were used to extract compounds from pomegranate peel with 60 % solvent extracts exhibited the strongest free‐radical scavenging activities among the three solvents used. Nine most consistently appeared chromatographic peaks were identified, namely gallic acid, gallagyl‐hexoside (punicalin), digalloly‐hexoside, galloyl‐HHDP‐hexoside, bis‐HHDP‐hexoside (pedunculagin I), punicalagin‐I, punicalagin‐II, granatin B, ellagic acid. Punicalagin was identified as the major component contributing to the potent free‐radical scavenging activities. All compounds except gallic acid and ellagic acid could directly react with the free‐radicals and thus exhibiting free‐radical scavenging activities. Pomegranate peel ellagitannins (PETs, mainly containing punicalagin), pomegranate peel ethyl acetate extracts (PPEEs, mainly containing granatin B and punicalagin), and 20% aqueous methanol extracts (M20, mainly containing punicalin, digalloly‐hexoside, galloyl‐HHDP‐hexoside, and pedunculagin I) were prepared and their anti‐inflammatory and anti‐colorectal cancer activities were examined in RAW 264.7 and HT‐29 colorectal cancers cells. PPEEs exhibited both superior anti‐inflammatory and anticolorectal cancer activities. Anti‐HT‐29 cancer cell and anti‐intestinal mucositis activities of PETs, PPEEs, and M20 were further examined in xenograft nude mice and chemotherapy 5‐FU treated rats. Consistent with the in vitro results, in vivo results showed that PPEEs exhibited the strongest anti‐HT‐29 cancer cell and anti‐intestinal mucositis activities. Cell death mechanisms of PPEEs on HT‐29 cells were further examined. PPEEs induced dose‐ and time‐dependent apoptosis in HT‐29 cells as shown by Annexin V‐FICT and PI double staining, together with western blotting of cleaved caspase 3, 8, and 9. Interestingly, autophagy was inhibited. Cell cycle analysis revealed that the cells were arrested in S phase. Moreover, ROS production was dose‐dependently stimulated by PPEEs. Anti‐oxidant N‐acetyl‐L‐cysteine pre‐incubation ameliorated PPEEs‐induced ROS stress, apoptosis, and cell cycle arrest. Collectively, the cell death might be initiated through ROS mediated autophagy inhibition, cell cycle arrest and apoptosis induction. Moreover, PPEEs enhanced cytotoxicity and apoptosis‐induction capability of 5‐FU against HT‐29 cells. Taken together, the results have provided evidence for developing PPEEs as a potential therapeutic agent for colorectal cancer and 5‐FU‐induced intestinal mucositis.Support or Funding InformationThis study was supported financially by Seed Fund for Translational and Applied Research (Project code: 201511160027) and Seed Fund for Basic Research (Project code: 201611159232) of The University of Hong Kong.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Reduced-gliotoxin induces ROS-mediated anoikis in human colorectal cancer cells.

Reduced-gliotoxin is a small molecule derived from the secondary metabolites of marine fungi; compared to other gliotoxin analogues, it exhibits potent anticancer effects. However, the molecular basis of the death of colorectal cancer (CRC) cells induced by reduced-gliotoxin is unclear. Thus, the aim of this study was to investigate the potency of reduced-gliotoxin against CRC cells and to elucidate the underlying mechanisms. Cell morphology, flow cytometric analysis and western bolt analysis were performed to examine the functions and mechanisms of cell death induced by reduced-gliotoxin. Our findings demonstrated that reduced-gliotoxin triggered rapid cell detachment and induced anoikis in CRC cells. Mechanistically, our data indicated that the anoikis induced by reduced-gliotoxin was associated with the disruption of integrin-associated cell detachment and multiple signaling pathways. Furthermore, reduced-gliotoxin induced the excessive production of reactive oxygen species (ROS) and the disruption of mitochondrial membrane potential (MMP), resulting in the activation of both endogenous and exogenous apoptotic pathways and eventually, in the apoptosis of CRC cells. The blockage of ROS generation with N-acetylcysteine(NAC) attenuated the anoikis induced by reduced-gliotoxin. Taken together, these results suggest that reduced-gliotoxin may prove to be a potential candidate in the treatment of CRC.

Non-Thermal Plasma Induced Immunogenic Cell Death In Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is a lethal diagnosis, with a 5-year survival rate of approximately 7%. PDAC often presents late, when the only curative option, surgery, in no longer viable. Even after curative surgery in patients with resectable disease, early recurrence and disease progression often occur. Non-thermal plasma therapy represents a novel, non-invasive, immunotherapeutic approach for management of PDAC. We have previously reported plasma induced immunogenic cell death (ICD) of CT-26 colorectal cancer cells in vitro. Moreover, we have shown, that a whole-cell vaccine produced by in vitro treatment of cancer cells with plasma has resulted in protective immunity in a mouse model of colorectal cancer. Here we tested the ability of plasma to induce ICD in a radiation and multi-drug resistant human epithelial pancreatic adenocarcinoma cell line, Panc-02, with a nanosecond pulsed dielectric barrier discharge (nspDBD). We report (Figure 1) decreased cell viability in a dose-dependent fashion in vitro. Additionally, ICD, as evidenced by increased expression of Calreticulin (CRT), a damage associated molecular pattern (DAMP) protein, is shown in Table 1. Mitoxantrone (MTX) is a candidate positive control for ICD in the Panc-02 cell line with a 45-fold increase in CRT expression associated with an approximate 20-25% decrease in cell viability. Cisplatin resulted in a 2-fold increase in CRT expression and an approximate 20-25% decrease in cell viability. The cell viability results were dose-independent for both MTX and Cisplatin. Vaccination results of C57B/6 mice with plasma treated Panc-02 cells will be presented. Download : Download high-res image (212KB) Download : Download full-size image Figure 1: Plasma Induced Panc-02 cell viability via PI assay.

The small molecule AU14022 promotes colorectal cancer cell death via p53-mediated G2/M-phase arrest and mitochondria-mediated apoptosis.

The p53 tumor suppressor plays critical roles in cell cycle regulation and apoptotic cell death, with its activation capable of sensitizing cancer cells to radiotherapy or chemotherapy. To identify small molecules that induce apoptosis via increased p53 transcriptional activity, we used a novel in-house library containing 96 small-molecule compounds. Using a cell-based screening method with a p53-responsive luciferase-reporter assay system involving benzoxazole derivatives, we found that AU14022 administration significantly increased p53 transcriptional activity in a concentration-dependent manner. Treatment with AU14022 increased p53 protein expression, p53 Ser15 phosphorylation, p53-mediated expression of downstream target genes, and apoptosis in p53-wild-type HCT116 human colon cancer cells, but not in p53-knockout HCT116 cells. Additionally, p53-wild-type HCT116 cells treated with AU14022 exhibited mitochondrial dysfunction, including modulated expression of B-cell lymphoma-2 family proteins and cytochrome c release. Combination treatment with AU14022 and ionizing radiation (IR) synergistically induced apoptosis as compared with IR or AU14022 treatment alone, with further investigation demonstrating that cell cycle progression was significantly arrested at the G2/M phase following AU14022 treatment. Furthermore, in a mouse p53-wild-type HCT116 colon cancer xenograft model, combined treatment with AU14022 and IR inhibited tumor growth more effectively than radiation alone. Therefore, AU14022 treatment induced apoptosis through p53-mediated cell cycle arrest involving mitochondrial dysfunction, leading to enhanced radiosensitivity in colon cancer cells. These results provide a basis for further assessments of AU14022 as a promising anticancer agent.

Up-regulation of UVRAG by HDAC1 Inhibition Attenuates 5FU-induced Cell Death in HCT116 Colorectal Cancer Cells.

The ultraviolent irradiation resistance-associated gene (UVRAG), a component of the Beclin 1/autophagy-related 6 complex, regulates the autophagy initiation step and functions in the DNA-damage response. UVRAG is frequently mutated in various cancer types, and mutations of UVRAG increase sensitivity to chemotherapy by impairing DNA-damage repair. In this study, we addressed the epigenetic regulation of UVRAG in colorectal cancer cells. UVRAG expression was increased in cells treated with histone deacetylase (HDAC) inhibitors, such as valproic acid and suberoylanilide hydroxamic acid. Down-regulation of HDAC1 enhanced UVRAG expression in colorectal cancer cells. In addition, both chemical and genetic inhibition of HDAC1 reduced the activation of caspase-3 and cytotoxicity in 5-fluorouracil (5FU)-treated cancer cells. In contrast, UVRAG overexpression inhibited caspase activation and cell death in 5FU-treated cells. Taken together, our findings suggest that up-regulation of UVRAG by HDAC1 inhibition potentiates DNA-damage-mediated cell death in colorectal cancer cells.

Bone marrow-derived mesenchymal stromal cells promote colorectal cancer cell death under low-dose irradiation

Background:Radiotherapy remains one of the cornerstones to improve the outcome of colorectal cancer (CRC) patients. Radiotherapy of the CRC not only help to destroy cancer cells but also remodel the tumour microenvironment by enhancing tumour-specific tropism of bone marrow-derived mesenchymal stromal cell (BM-MSC) from the peripheral circulation. However, the role of local MSCs and recruited BM-MSC under radiation were not well defined. Indeed, the functions of BM-MSC without irradiation intervention remained controversial in tumour progression: BM-MSC was previously shown to modulate the immune function of major immune cells, resulting in an impaired immunological sensitivity and to induce an increased risk of tumour recurrence. In contrast, it could also secrete various cytokines and possess anticancer effect.Methods:Three co-cultivation modules, 3D culture modules, and cancer organoids were established. The induction of cytokines secretion in hBM-MSCs after irradiation was analysed by ELISA array and flow cytometry. AutoMac separator was used to separate hBM-MSC and CRC automatically. Cells from the co-cultured group and the control group were then irradiated by UV-C lamp and X-ray. Proliferation assay and viability assay were performed.Results:In this study, we show that BM-MSCs can induce the EMT progression of CRC cells in vitro. When irradiated with low doses of ultraviolet radiation and X-rays, BM-MSCs show an anti-tumour effect by secreting certain cytokine (TNF-α, IFN-γ) that lead to the inhibition of proliferation and induction of apoptosis of CRC cells. This was further verified in a 3D culture model of a CRC cell in vitro. Furthermore, irradiation on the co-culture system induced the cleavage of caspase3, and attenuated the phosphorylation of phosphatidylinositol 3-kinase (PI3K)/AKT and extracellular signal-regulated kinase in cancer cells. The signal pathways above might contribute to the cancer cell death.Conclusions:Taken together, we show that BM-MSC can potentially promote the effect of radiotherapy in CRC.