Multidrug Resistant Colon Cancer Research Articles

Isoliensinine augments the therapeutic potential of paclitaxel in multidrug-resistant colon cancer stem cells and induced mitochondria-mediated cell death.

Previously we have reported the isoliensinine (ISO) potentates the therapeutic potential of cisplatin in cisplatin resistant colorectal cancer stem cells. The present study evaluates the chemo-sensitizing potential of the combinatorial regimen of ISO and Paclitaxcel (PTX) on multidrug-resistant (MDR)-HCT-15 cells to reduce the dose requirement of both ISO and PTX. The results of the present study suggest that treatment with the combinatorial regimen of ISO and PTX enhanced the cytotoxic effect with resultant increase in apoptosis in MDR-HCT-15 cells as evident from the altered cellular morphology, G2/M cell cycle arrest, propidium iodideuptake, Annexin V, increased intracellular Ca2+ accumulation, decreased mitochondrial membrane potential, diminished ATP production, PARP-1 cleavage, altered expression of ERK1/2, and apoptotic proteins. Treatment with combinatorial regimen of ISO and PTX also modulated the expression of the transcription factors SOX2, OCT4 which determine the stemness of cancer cells. Thus, results of the present study suggest that ISO and PTX combination regimen induces apoptosis in MDR-HCT-15 in a synergistic manner.

Abstract LB131: Shortening of mRNA 3′ untranslated region mediates simultaneous overexpression of ABCG2 and CD133 in putative cancer stem cells

Abstract Aim: We investigated the mechanisms driving the concomitant overexpression of ABCG2 and CD133 in cancer stem cells (CSCs) and examined the effect of modulating the underlying machinery on anticancer drug response. Background: Multidrug resistance is a major obstacle to chemotherapy. It is usually associated with increased efflux of anticancer drugs by ATP-binding cassette transporters including ABCG2. ABCG2 is also a key survival factor for CSCs, which lead to recurrence. CSCs have been identified in patient tumor specimens and in cancer cell lines. The CSC hypothesis suggests that anticancer therapies have to target and destroy all residual CSCs in order to produce a durable response. CD133 is a well-characterized stem cell biomarker and it plays crucial role in the self-renewal and tumorigenesis of CSCs. Importantly, ABCG2 and CD133 are co-expressed in CSCs, yet the mechanism(s) driving their concomitant high expression remain elusive. mRNA polyadenylation is a key step regulating the maturation of eukaryotic mRNAs by adding a poly A tail to their 3' ends. Alternative polyadenylation, commonly observed in cancer cells, is a phenomenon responsible for the production of multiple mRNA isoforms bearing different length of 3'-untranslated region (3'UTR). Method: Sub-population of cancer cells from three ABCG2-overexpressing multidrug resistant colon cancer cell lines were sorted by Hoechst dye exclusion or CSC markers (DCLK1+LGR5+BMI1+), respectively, as side population (SP) and putative CSC cells. Expression of ABCG2, CD133, and a few master mRNA polyadenylation trans-acting regulators was compared in SP (or CSC) versus non-SP (or non-CSC) cells. Altered 3'UTR isoforms of ABCG2 and CD133 were examined by 3'RACE assay and 3'UTR isoform specific PCR. The anticancer activity of common chemotherapeutic drugs was examined after genetic modulation of the key mRNA polyadenylation regulators identified. Result: The mRNA stability and expression of both ABCG2 and CD133 were found to be remarkably higher in SP cells (or CSC cells) than in non-SP cells (or non-CSC cells). By 3'RACE assay, shortening of ABCG2 and CD133 3'UTR was evidenced in SP (and CSC) cells but not in non-SP (and non-CSC) cells, thereby increasing mRNA stability and/or translation by escaping the repression from regulatory elements found on long mRNA 3'UTR. Expression of two master mRNA polyadenylation regulators (PABPN1 and CSTF2) was found to be remarkably lower and higher, respectively, in SP (and CSC) than in non-SP (and non-CSC) cells, presumably allowing the preferential utilization of proximal 3'UTR to mediate high expression of ABCG2 and CD133. Genetic silencing of CSTF2 was shown to enhance the anticancer effect of SN-38 and reduce the colony formation capacity of CSC cells. Conclusion: A novel mRNA 3'UTR shortening machinery was shown to mediate the simultaneous overexpression of ABCG2 and CD133 in SP and CSC cells. It may represent useful drug target for circumvention of resistance and eradication of CSCs. Citation Format: Kenneth K.W. To. Shortening of mRNA 3′ untranslated region mediates simultaneous overexpression of ABCG2 and CD133 in putative cancer stem 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 LB131.

Study on reversal of ABCB1 mediated multidrug resistance in Colon cancer by acetogenins: An in-silico approach

Multi-Drug Resistance (MDR) exerted by tumor cells is majorly due to the overexpression of ATP Binding cassette transporters such as ABCB1/P-glycoprotein (P-gp). Annonaceous acetogenins (AGEs) exert anticancer activity by strongly inhibiting NADH oxidase of cancer cells. The present in silico study aims at screening a potent MDR inhibitor among acetogenins from the plant Annona muricata. Twenty-four AGEs were selected and screened for their pharmacokinetic properties. An inward facing conformation of P-gp is required for understanding the interaction of AGEs at the drug binding region and hence the human P-gp protein was modeled. The selected compounds were then docked with the ATP binding site and the drug binding site of modeled human P-gp. Annonacin A.1, Annohexocin.1 and Annomuricin E.1 docked better with high MM/GBSA dG binding in the drug binding region as compared with the conventional drugs. These compounds had a better docking score as compared with control inhibitor drugs at the ATP binding region. The complexes were subjected to MD simulation and Annonacin A was stable throughout the simulation period. Therefore, Annonacin A might act as a competitive inhibitor for the chemo drugs for binding at the drug binding region of P-gp. Hence it is capable of decreasing the efflux of chemo drugs out of the cells by P-Glycoprotein/ABCB1/MDR1. With this computational study, it is concluded that this compound might potentially reverse MDR, and hence can be taken forward for validation studies. Communicated by Ramaswamy H. Sarma

Poziotinib Inhibits the Efflux Activity of the ABCB1 and ABCG2 Transporters and the Expression of the ABCG2 Transporter Protein in Multidrug Resistant Colon Cancer Cells.

Simple SummaryGlobally, colorectal cancer (CRC) is a leading cause of cancer deaths and chemotherapy, in combination with radiotherapy when appropriate, is used to treat the majority of CRC patients. However, the acquisition or development of drug resistance can decrease, or even abolish, the efficacy of chemotherapy. ATP-binding cassette (ABC) transporters, particularly, the ABCB1 and ABCG2 transporter, are mediators of multidrug resistance (MDR) in certain types of cancer cells. The aim of our in vitro study was to determine if poziotinib can overcome MDR to certain chemotherapeutic drugs in colon cancer cells. Our results indicated that in MDR CRC cell lines, poziotinib inhibits the transport function of the ABCB1 and ABCG2 transporters, increasing the intracellular accumulation of certain anticancer drugs, and thus, their efficacy. Furthermore, poziotinib decreased the expression of the ABCG2 protein. Therefore, if our results can be translated to humans, they suggest that using poziotinib in combination with certain anticancer drugs may be of therapeutic benefit in colorectal cancer patients.Colorectal cancer (CRC) is a leading cause of cancer deaths in the United States. Currently, chemotherapy is a first-line treatment for CRC. However, one major drawback of chemotherapy is the emergence of multidrug resistance (MDR). It has been well-established that the overexpression of the ABCB1 and/or ABCG2 transporters can produce MDR in cancer cells. In this study, we report that in vitro, poziotinib can antagonize both ABCB1- and ABCG2-mediated MDR at 0.1–0.6 μM in the human colon cancer cell lines, SW620/Ad300 and S1-M1-80. Mechanistic studies indicated that poziotinib increases the intracellular accumulation of the ABCB1 transporter substrates, paclitaxel and doxorubicin, and the ABCG2 transporter substrates, mitoxantrone and SN-38, by inhibiting their substrate efflux function. Accumulation assay results suggested that poziotinib binds reversibly to the ABCG2 and ABCB1 transporter. Furthermore, western blot experiments indicated that poziotinib, at 0.6 μM, significantly downregulates the expression of the ABCG2 but not the ABCB1 transporter protein, suggesting that the ABCG2 reversal effect produced by poziotinib is due to transporter downregulation and inhibition of substrate efflux. Poziotinib concentration-dependently stimulated the ATPase activity of both ABCB1 and ABCG2, with EC50 values of 0.02 μM and 0.21 μM, respectively, suggesting that it interacts with the drug-substrate binding site. Molecular docking analysis indicated that poziotinib binds to the ABCB1 (−6.6 kcal/mol) and ABCG2 (−10.1 kcal/mol) drug-substrate binding site. In summary, our novel results show that poziotinib interacts with the ABCB1 and ABCG2 transporter, suggesting that poziotinib may increase the efficacy of certain chemotherapeutic drugs used in treating MDR CRC.

Abstract 5264: Selective toxicity of MX-106-4C, a survivin inhibitor, in P-glycoprotein-mediated multidrug resistant colon cancer

Abstract One of the major challenges in colon cancer chemotherapy is multidrug resistance (MDR), which is typically mediated by the overexpression of ATP-binding cassette (ABC) transporters, particularly P-glycoprotein (P-gp, ABCB1, MDR1). A number of P-gp inhibitors have been developed, however, none of these compounds have improved chemotherapeutic efficacy due to undesirable pharmacokinetic profiles or adverse effects, resulting in limited clinical success. Therefore, alternative approaches are urgently needed to circumvent MDR cancer. In previous study, a series of synthesized analogs of MX-106, as anti-cancer drugs targeting survivin, exhibited collateral sensitivity (CS) effect to P-gp overexpressing MDR colon cancer cells as well as ABCB1 gene transfected cells, reflected by more than 10-fold cytotoxic effect in P-gp positive MDR cell lines compared to drug sensitive cell lines. Among the analogs, MX-106-4C was identified as the leading compound with the most potent selective toxicity to P-gp overexpressing cells. MX-106-4C-induced CS effect was observed in both intrinsic and acquired P-gp overexpressing colon cancer cells, which was only partially reversed with the presence of a P-gp inhibitor. Nevertheless, this CS effect was abolished in ABCB1-knockout cells, indicating that the selective cytotoxicity was P-gp expression dependent, but only partially related to P-gp function. Furthermore, we found that MX-106-4C did not significantly affect P-gp ATPase activity or drug accumulation and efflux in P-gp-overexpressing cells. In P-gp overexpressing colon cancer cells, short-term (up to 72 h) incubation of MX-106-4C significantly down regulated P-gp expression at transcriptional level but not protein level, whereas long-term (14 d) incubation of MX-106-4C significantly down regulated P-gp protein expression and re-sensitized MDR colon cancer cells to doxorubicin. These findings suggested an indirect interaction and regulation between MX-106-4C and P-gp. Further study revealed that the selective cytotoxic effects of MX-106-4C were associated with cell cycle arrest at G1 phase and apoptosis through the downregulation of CDK4. Overall, this study demonstrates that MX-106-4C selectively kills P-gp positive MDR colon cancer cells and indirectly regulates P-gp, which provides a clue for CS compound design and a novel strategy to obviate P-gp-mediated colon cancer MDR by re-sensitizing heterogeneous tumors with CS agents. Citation Format: Zi-Ning Lei, Zhongzhi Wu, Qiu-Xu Teng, Min Xiao, Wei Li, John N. Wurpel, Zhe-Sheng Chen. Selective toxicity of MX-106-4C, a survivin inhibitor, in P-glycoprotein-mediated multidrug resistant colon cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5264.

Abstract 6326: Global changes in alternative mRNA polyadenylation profile promote the development of multidrug resistance (MDR) to chemotherapy in colon cancer

Abstract Aim: The study investigated the global changes in mRNA alternative polyadenylation (APA) profile associated with MDR development in colon cancer cells. Background: Polyadenylation is a crucial step needed for the maturation of eukaryotic mRNAs. APA refers to the production of different mRNA isoforms bearing varying length of 3'-untranslated region (3'UTR), where key RNA regulatory elements are found. APA usually eliminates the distal end of the 3'UTR, thus allowing the escape from the predominantly repressive effect from longer 3'UTR. Colon cancer is a leading cause of cancer-related death worldwide. MDR, which is usually associated with overexpression of ATP-binding cassette efflux transporters including ABCG2, is a major unresolved obstacle to successful cancer chemotherapy. We have previously reported that ABCG2 mRNA is more stable in MDR colon cancer cells. Interestingly, the increase in mRNA stability is tied to a missing microRNA binding site (miR-519c) due to the shortening of ABCG2 mRNA 3'UTR in drug resistant cells. Thus microRNA-mediated mRNA degradation and/or protein translation block are relieved, thereby leading to ABCG2 overexpression. A remarkable feature of 3'UTR APA is that it can be regulated globally, thus simultaneously affecting numerous mRNA transcripts. While ABCG2 mRNA 3'UTR is shortened in MDR colon cancer cells, the global changes in APA profile of other transcripts during the course of MDR development have not been reported. Method: Differential APA signature and specific APA-regulated mRNAs were evaluated in two pairs of sensitive and MDR colon cancer cell lines by RNA sequencing. APA profiles were assessed from the RNA-Seq datasets by using the bioinformatics tool “DaPars”. Altered 3'UTR isoforms of a few differentially expressed mRNAs were validated by 3'RACE assay and 3'UTR isoform specific PCR analysis. The genes were subjected to Gene Ontology (GO) analysis to identify relevant molecular pathways and biological processes. Result: APA profiles of two pairs of drug resistant ABCG2-overexpressing colon cancer cell line (S1-M1-80 & SW620 Flv100) versus their parental cells (S1 & SW620) were compared at the global scale from their sequenced RNA samples. By GO analysis using the PANTHER Platform, mRNA transcripts exhibiting 3'UTR shortening in S1-M1-80 and SW620 Flv100 versus their respective parental cells were enriched in biological processes including “transport and catabolism”, “cell growth and death”, and “cell motility” by GO terms. Genes ranked at the top of the list are mostly direct or indirect targets of the Wnt-β-catenin signaling pathway. Wnt signaling is a crucial pathway for mediating metastasis and therapeutic resistance. Conclusion: Global changes of APA in gene regulation may contribute to the development of MDR in colon cancer. Further investigation is warranted to elucidate the mechanisms leading to APA. Modulation of the APA machinery may help eradicate MDR cancer cells. Citation Format: Kenneth KW To. Global changes in alternative mRNA polyadenylation profile promote the development of multidrug resistance (MDR) to chemotherapy in colon cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6326.

Enhanced absorption, and efficacy of oral self-assembled paclitaxel nanocochleates in multi-drug resistant colon cancer.

Chemotherapy in drug-resistant cancers remains a challenge. Owing to associated poor bioavailability, oral administration of hydrophobic anticancer drugs like paclitaxel has been quite challenging, with the scenario being further complicated by Pgp efflux in drug-resistant tumours. We developed a novel nanocochleates (CPT) system encapsulating paclitaxel (PTX) to treat resistant colon cancer by oral administration. PTX encapsulated nanocochleates (PTX-CPT), made up of phosphatidylserine in size range of 350-600nm with -20±5.2mV zeta potential were protected from degradation at acidic gastric pH and showed sustained PTX release over 48h under intestinal pH condition. In vitro cytotoxicity studies on HCT-116 & HCT-15 cells (multi-drug resistant) established IC50 value of <10 and 69nM, respectively, which was significantly lower when compared to commercial Taxol formulation. Further, the in vivo efficacy with five oral doses of 30mg/kg PTX-CPT in an HCT-15 drug-resistant colon cancer xenograft mouse model showed more than 25 fold reduction in the tumour growth inhibition as compared to intravenous Taxol which showed just 1.94% inhibition. Interestingly, PTX-CPT treated mice also showed significantly lower proliferation index and microvessel density when compared to Taxol treated mice. Nanocochleates showed lower toxicity with at LD-50 value greater than 300mg/kg as described in OECD 423 guideline. The enhanced efficacy of PTX-CPT speculated due to its internalization by active endocytosis, ability to escape Pgp efflux, and due to a combined effect of the pro-apoptotic and antiangiogenic role. Taken together, the results suggested the PTX-CPT a promising strategy for efficiently treating drug-resistant colon cancer orally.

A pH/ROS cascade-responsive and self-accelerating drug release nanosystem for the targeted treatment of multi-drug-resistant colon cancer

The efficacy of chemotherapeutic agents for colon cancer treatment is limited by multidrug resistance (MDR) and insufficient intracellular release of the administered nanomedicine. To overcome these limitations, we constructed a pH/ROS cascade-responsive and self-accelerating drug release nanoparticle system (PLP-NPs) for the treatment of multidrug-resistant colon cancer. The PLP-NPs comprised a reactive oxygen species (ROS)-sensitive polymeric paclitaxel (PTX) prodrug (DEX-TK-PTX), a pH-sensitive poly(l-histidine) (PHis), and beta-lapachone (Lapa), a ROS-generating agent. We found that PLP-NPs could accumulate in tumor tissue through enhancement of the permeability and retention (EPR) effect, and were subsequently internalized by cancer cells via the endocytic pathway. Within the acidic endo-lysosomal environment, PHis protonation facilitated the escape of the PLP-NPs from the lysosome and release of Lapa. The released Lapa generated a large amount of ROS, consumed ATP, and downregulated P-glycoprotein (P-gp) production through the activity of NQO1, an enzyme that is specifically overexpressed in tumor cells. In addition, the generated ROS promoted the release of PTX from DEX-TK-PTX to kill cancer cells, while ATP depletion inhibited P-gp-mediated MDR. In vitro and in vivo experiments subsequently confirmed that PLP-NPs induced tumor-specific cytotoxicity and overcame the MDR of colon cancer. Our findings indicate that the use of the PLP-NPs system represents a promising strategy to counter MDR in the treatment of colon cancer.

Abstract LB-263: miR-181a downregulation allows simultaneous overexpression of ABCG2 and induction of autophagy to mediate multidrug resistance in colon cancer

Abstract Aim: The study investigated the cross-talk between overexpression of multidrug resistance (MDR) transporter ABCG2 and induction of autophagy in mediating drug resistance in colon cancer. We also studied the simultaneous inhibition of ABCG2 and autophagy as a novel strategy to circumvent MDR. Background: Colon cancer is a leading cause of cancer-related death worldwide. MDR, which is usually associated with overexpression of ATP-binding cassette efflux transporters including ABCG2, is a major unresolved obstacle to successful cancer chemotherapy in colon cancer. Recent investigations have shown that autophagy is also involved in the development of MDR. Autophagy is an adaptive and survival mechanism in response to stress and starvation in order to maintain cellular homeostasis. Interestingly, ABCG2 overexpression was found to enhance autophagy to promote cell survival in response to various stress. Co-inhibition of ABCG2 and autophagy might exert a synergistic cancer killing effect to help eradicate MDR cancer cells. Our team has recently reported the upregulation of ABCG2 in tumor specimens, relative to adjacent normal colon epithelial cells, obtained from colon cancer patients unresponsive to 5-fluorouracil-based chemotherapy. Interestingly, this is coincident with a remarkable downregulation of miR-181a, which is also known to target and repress a major autophagy regulator ATG5. Therefore, we hypothesize that miR-181a may form a mechanistic link between ABCG2 overexpression and autophagy induction to mediate MDR. Method: Regulation of ABCG2 and ATG5 in MDR colon cancer cells after ectopic overexpression or genetic silencing of miR-181a was investigated. ABCG2 and ATG5 expression was measured by qPCR and Western blot analysis. Functional consequence of ABCG2 regulation was evaluated by measuring efflux and anticancer activity of ABCG2 substrate anticancer drugs. Autophagic flux was monitored by LC3 turnover and selective autophagy substrate p62 degradation assay. Result: In ABCG2-overexpressing MDR colon cancer cell lines, miR-181a expression was found to be remarkably reduced in the resistant sublines relative to the parental cells. Ectopic overexpression of miR-181a was found to downregulate both ABCG2 and ATG5, which also lead to reduced drug efflux, inhibition of autophagic flux and enhanced anticancer activity of both ABCG2 substrate (SN-38) and non-substrate (oxaliplatin) anticancer drugs. We screened a compound library and identified autophinib as a compound capable of inhibiting both ABCG2 and autophagy. At non-toxic concentration, autophinib was found to potentiate anticancer activity in MDR colon cancer cells. Conclusion: Our study revealed the causal and complementary relationship between ABCG2 overexpression and autophagy induction in the development of MDR in colon cancer. miR-181a may represent a new molecular target for MDR circumvention. Co-inhibition of ABCG2 and autophagy was found to circumvent drug resistance in MDR cells. Note: This abstract was not presented at the meeting. Citation Format: Kenneth KW To. miR-181a downregulation allows simultaneous overexpression of ABCG2 and induction of autophagy to mediate multidrug resistance in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-263.

Abstract 2116: Cariprazine is a potent chemo-adjuvant against multidrug resistant colon and lung cancer

Abstract Multidrug resistance (MDR) is considered a continuing clinical problem, reducing the efficacy of cancer chemotherapy. The over expression of the ATP - binding cassette (ABC) family G2 (ABCG2) transporter is considered to be one of the main mechanisms responsible for mediating MDR in cancer. Molecular modeling studies have shown the significant affinity and binding mode of cariprazine, a dopamine D3-preferring D2/D3 receptor partial agonist, to the drug binding cavity of ABCG2 with a Glide XP score of the -5.761. Therefore, in this in vitro study, we determined the effect of cariprazine in increasing the efficacy of MX (an ABCG2 substrate) in the ABCG2 overexpressing cancer cell lines H460-MX20 and S1M1-80. Also, studies conducted to determine whether cariprazine is a reversal compound that specifically targets the ABCG2 protein. Alone, cariprazine, at concentrations up to 20 μM, did not significantly decrease cell viability. Cariprazine, at concentrations ranging from 1 to 20 μM, did not significantly alter the cytotoxicity of mitoxantrone (MX) in the parental non-small cell cancer cell line, H460 and colon cancer cell S1. However, cariprazine (1-20 μM) significantly enhanced the efficacy of ABCG2 substrate antineoplastic drug MX in the ABCG2- overexpressing MDR cell line, H460-MX20 and S1M1-80, by reducing the resistance fold from 28 to 1 and from 93 to 1.33, respectively. The intracellular accumulation of rhodamine 123 in the ABCG2-overexpressing cells, S1M1-80 was significantly increased by the addition of cariprazine, in a concentration-dependent manner (1 - 20 μM). Interestingly, 10 or 20 μM of cariprazine significantly decreased the expression levels of the ABCG2 protein in the colon and lung cancer cell lines, suggesting that cariprazine inhibits both the function and expression of ABCG2 transporters at non-toxic concentrations. Overall, our results suggest that cariprazine, via several distinct mechanisms, can re-sensitize resistant cancer cells to mitoxantrone. Citation Format: Noor Hussein, Angelique Nyinawbera, Haneen Amawi, Amit Tiwari. Cariprazine is a potent chemo-adjuvant against multidrug resistant colon and lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2116.

Retraction: Sinomenine Sensitizes Multidrug-Resistant Colon Cancer Cells (Caco-2) to Doxorubicin by Downregulation of MDR-1 Expression.

Retraction: Sinomenine Sensitizes Multidrug-Resistant Colon Cancer Cells (Caco-2) to Doxorubicin by Downregulation of MDR-1 Expression.

Reversal of Multidrug Resistance in Human Colon Cancer and Human Leukemia Cells by Three Plant Extracts and Their Major Secondary Metabolites.

Background: We studied the effect of three plant extracts (Glycyrrhiza glabra, Paeonia lactiflora, Eriobotrya japonica) and six of their major secondary metabolites (glycyrrhizic acid, 18β glycyrrhetinic acid, liquiritigenin, isoliquiritigenin, paeoniflorin, ursolic acid) on the multidrug resistant human colon cancer cell line Caco-2 and human leukemia cell line CEM/ADR 5000 as compared to the corresponding sensitive cell line CCRF-CEM, and human colon cancer cells HCT-116, which do not over-express ATP-binding cassette (ABC) transporters. Methods: The cytotoxicity of single substances in sensitive and resistant cells was investigated by MTT assay. We also applied combinations of extracts or single compounds with the chemotherapeutic agent doxorubicin or doxorubicin plus the saponin digitonin. The intracellular retention of the ABC transporter substrates rhodamine 123 and calcein was examined by flow cytometry to explore the effect of the substances on the activity of ABC transporters P-glycoprotein and MRP1. Real-time PCR was applied to analyse the gene expression changes of ABCB1, ABCC1, caspase 3, caspase 8, AhR, CYP1A1, and GSTP1 in resistant cells under the treatment of the substances. Results: All the substances moderately inhibited cell growth in sensitive and resistant cells to some degree. Whereas ursolic acid showed IC50 of 14 and 22 µM in CEM/ADR 5000 and Caco-2 cells, respectively, glycyrrhizic acid and paeoniflorin were inactive with IC50 values above 400 μM. Except for liquiritigenin and isoliquiritigenin, all the other substances reversed MDR in CEM/ADR 5000 and Caco-2 cells to doxorubicin. Ue, ga, 18ga, and urs were powerful reversal agents. In CEM/ADR 5000 cells, high concentrations of all the substances, except Paeonia lactiflora extract, increased calcein or rhodamine 123 retention in a dose-dependent manner. In Caco-2 cells, all the substances, except liquiritigenin, retained rhodamine 123 in a dose-dependent manner. We also examined the effect of the plant secondary metabolite (PSM) panel on the expression of ABCB1, ABCC1, caspase 3, caspase 8, AhR, CYP1A1, and GSTP1 genes in MDR cells. Conclusions: The extracts and individual PSM could reverse MDR in CEM/ADR 5000 and Caco-2 cells, which overexpress ABC transporters, in two- and three-drug combinations. Most of the PSM also inhibited the activity of ABC transporters to some degree, albeit at high concentrations. Ue, ga, 18ga, and urs were identified as potential multidrug resistance (MDR) modulator candidates, which need to be characterized and validated in further studies.

Bufalin-loaded vitamin E succinate-grafted-chitosan oligosaccharide/RGD conjugated TPGS mixed micelles demonstrated improved antitumor activity against drug-resistant colon cancer.

BackgroundMultidrug resistance (MDR) is the major reason for the failure of chemotherapy in colon cancer. Bufalin (BU) is one of the most effective antitumor active constituents in Chansu. Our previous study found that BU can effectively reverse P-glycoprotein (P-gp)-mediated MDR in colon cancer. However, the clinical application of BU is limited due to its low solubility in water and high toxicity. In the present study, a multifunctional delivery system based on vitamin-E- succinate grafted chitosan oligosaccharide (VES-CSO) and cyclic (arginine-glycine-aspartic acid peptide) (RGD)-modified d-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) was prepared by emulsion solvent evaporation method for targeted delivery of BU to improve the efficacy of drug-resistant colon cancer therapy.MethodsThe cytotoxicity of BU-loaded micelles against drug-resistant colon cancer LoVo/ADR and HCT116/LOHP cells was measured by CCK-8 assay. The cellular uptake, Rho123 accumulation, and cell apoptosis were determined by flow cytometry. The expression of apoptosis-related protein and P-gp was measured by Western blot assay. The antitumor activity of BU-loaded micelles was evaluated in LoVo/ADR-bearing nude mice.ResultsBU-loaded VES-CSO/TPGS-RGD mixed micelles (BU@VeC/T-RGD MM) were 140.3 nm in diameter with zeta potential of 8.66 mV. The BU@VeC/T-RGD MM exhibited good stability, sustained-release pattern, higher intracellular uptake, and greater cytotoxicity in LoVo/ADR cells. Furthermore, the mechanisms of the BU@VeC/T-RGD MM to overcome MDR might be due to enhanced apoptosis rate and P-gp efflux inhibition. Subsequently, in vivo studies confirmed an enhanced therapeutic efficiency and reduced side effects associated with BU@VeC/T-RGD MM compared with free BU, owing to the enhanced permeation and retention effect, improved pharmacokinetic behavior, and tumor targeting, which lead to MDR-inhibiting effect in LoVo/ADR-bearing nude mice.ConclusionOur results demonstrated that VeC/T-RGD MM could be developed as a potential delivery system for BU to improve its antitumor activity against drug-resistant colon cancer.

Regorafenib antagonizes BCRP-mediated multidrug resistance in colon cancer

Overexpression of breast cancer resistance protein (BCRP) has been shown to produce multidrug resistance (MDR) in colon cancer, leading to major obstacles for chemotherapy. In this study, we evaluated the effect of regorafenib, an oral multi-kinase inhibitor, in inhibiting BCRP-mediated MDR in silico, in vitro and in vivo. We found that regorafenib significantly sensitized MDR colon cancer cells to BCRP substrates by increasing their intracellular accumulation. There are no significant changes in the expression level or the subcellular distribution of BCRP in the cells exposed to regorafenib. Investigation of the mechanism revealed that regorafenib stimulated BCRP ATPase activity. Our induced-fit docking and molecular dynamics simulations suggested the existence of a strong and stable interaction between regorafenib and the transmembrane domain of human crystalized BCRP. In vivo tumor xenograft study revealed that the combination of regorafenib and topotecan exhibited synergistic effects on mitoxantrone-resistant S1-M1-80 xenograft tumors. In conclusion, our studies indicate that regorafenib would be beneficial in combating MDR in colon cancer.

Curcumin Co-Treatment Sensitizes Multi-Drug Resistant Ht29 Colon Cancer Cell Line

Colorectal cancer has been confirmed to be the third most dreadful cancer across the world. The latest reports show that the colorectal cancer is increasing in India at a high rate due to food habits, particularly consuming the foods with high fat content. Chemotherapy has been considered as a potential treatment in the control of a wide range of cancers recently, such as gastrointestinal cancers and so on. The scientists are looking to come up with new drugs to treat cancer by regulating the carcinogenesis with minimal toxicity. Curcumin is a phytochemical extracted from turmeric. It is highly effective product of the rhizomes of Curcuma longa L. (Zingiberaceae). It is also considered as a valuable drug for chemotherapeutic treatments in cancer. In case of animal studies, it has been clearly mentioned that curcumin controls carcinogenesis in various other organs as well as colon. Curcumin exhibits the capability to act as anti-mutagenic, anti-inflammatory drug as well. It has also been confirmed that the P-gp expression regulation is carried out by curcumin by inhibiting the COX-2 expression. Also, it has been clearly stated that curcumin down-regulates NF-kB pathway in various cell lines like Colo 205 colon cancer cell line etc. Multi Drug Resistance (MDR) plays a key role in case of the cancer cells to exhibit the resistance against the cytotoxicity of various chemotherapeutic drugs. This type of characteristics in cancer cells is developed due to low levels of the chemotherapeutic drug accumulated inside the cells during repeated exposure to the drug, showing the over-expression of P-glycoprotein (MDR-1). P-glycoprotein, a transporter protein, allows the cells to expel the wide range of chemotherapeutic drugs. The latest reports show that COX-2 expression and P-gp expression possess strong correlation with each other. In this research study, we generated the Multi-Drug Resistant HT29 Human colon cancer cells by treating the cells with increasing concentrations of a chemotherapeutic drug namely Doxorubicin Hydrochloride (DOX) and further we estimated the intracellular drug concentration in treated live as well as dead cells with the help of Doxorubicin Accumulation Assay. Also, the cell morphology change was studied in HT29 cells after every 24 hours of the DOX treatment separately as well as curcumin co-treatment (DOX+Curcumin). The curcumin co-treatment was carried out to observe the effect of curcumin on multi-drug resistant HT29 colon cancer cells and further study can be extended to investigate the various inflammatory genes controlled by NFkB at mRNA and protein level as well as the levels of cytokines which may get down-regulated by treatment. This study will help to completely understand the mechanism of reducing the effect of Multidrug Resistance (a unique property of cancer) by curcumin in case of colon cancer.

Curcumin Co-Treatment Sensitizes Multi-Drug Resistant Ht29 Colon Cancer Cell Line

Curcumin Co-Treatment Sensitizes Multi-Drug Resistant Ht29 Colon Cancer Cell Line

Vitexin induces apoptosis by suppressing autophagy in multi-drug resistant colorectal cancer cells.

Cancer treatment is limited due to the diverse multidrug resistance acquired by cancer cells and the collateral damage caused to adjacent normal cells by chemotherapy. The flavonoid compound vitexin exhibits anti-oxidative, anti-inflammatory and anti-tumor activity. This study elucidated the antitumor effects of vitexin and its underlying mechanisms in a multi-drug resistant human colon cancer cell line (HCT-116DR), which exhibits higher levels of multidrug-resistant protein 1 (MDR1) expression as compared with its parental cell line (HCT-116). Here, we observed that vitexin suppressed MDR-1 expression and activity in HCT-116DR cells and showed cytotoxic effect in HCT-116DR cells by inhibiting autophagy and inducing apoptosis in a concentration-dependent manner. Additionally, vitexin treatment caused cleavage of caspase-9 and caspase-3, and upregulated the expression of the pro-apoptotic proteins, BID and Bax. Moreover, the expression of autophagy-related proteins, such as ATG5, Beclin-1 and LC3-II, was markedly reduced by vitexin treatment. Furthermore, in vivo experiments showed that vitexin induced apoptosis and suppressed tumor growth in HCT-116DR xenograft model. These results revealed that vitexin induced apoptosis through suppression of autophagy in vitro and in vivo and provide insight into the therapeutic potential of vitexin for the treatment of chemo-resistant colorectal cancer.

Cryptotanshinone induces ROS-dependent autophagy in multidrug-resistant colon cancer cells

The development of novel chemotherapeutic agents is highly desired for colon cancer treatment, in particular for the multidrug-resistant cancer types. Cryptotanshinone (CTS), an active quinoid diterpene isolated from Salvia miltiorrhiza Bunge, was previously reported to induce autophagy in various colon cancer cell lines. However, its mechanisms of action have not been fully understood. The current study aims to explore the mechanisms by which CTS induces autophagy in a multidrug-resistant human colon cancer cell line SW620 Ad300. Using MTT assay, CTS at 10 μM exhibited no significant cytotoxicity on human normal colon fibroblasts CCD-18Co, but induced 45.67% and 48.35% cell death in SW620 and SW620 Ad300 cells, respectively. Further studies revealed that CTS induced weak apoptosis (9.37%) and significant caspase-independent cell death in SW620 Ad300 cells. In the same cell line, CTS also induced significant autophagy, which was found to promote cell death and to mediate the cytotoxicity of CTS in these multidrug-resistant cells. Moreover, activation of ROS-p38 MAPK-NF-κB signaling pathway was involved in autophagic cell death induced by CTS in SW620 Ad300 cells. Interestingly, our results also demonstrated a complementary relationship between CTS-induced apoptosis and autophagic cell death in SW620 Ad300 cells. Taken together, CTS induces autophagic cell death in SW620 Ad300 cells via the ROS-p38 MAPK-NF-κB signaling pathway, and it might be a potential candidate as a chemotherapeutic agent for the treatment of multidrug-resistant colon cancer.

Twist1 confers multidrug resistance in colon cancer through upregulation of ATP-binding cassette transporters.

Multidrug resistance is a major problem in colon cancer treatment. However, its molecular mechanisms remain unclear. Recently, the epithelial-mesenchymal transition (EMT) in anticancer drug resistance has attracted increasing attention. This study investigated whether vincristine treatment induces EMT and promotes multidrug resistance in colon cancer. The result showed that vincristine treatment increases the expression of several ATP-binding cassette transporters in invasive human colon adenocarcinoma cell line (HCT-8). Vincristine-resistant HCT-8 cells (HCT-8/V) acquire a mesenchymal phenotype, and thus its migratory and invasive ability are increased both in vitro and in vivo. The master transcriptional factors of EMT, especially Twist1, were significantly increased in the HCT-8/V cell line. Moreover, the ectopic expression of Twist1 increased the chemoresistance of HCT-8 cells to vincristine and increased the expression levels and promoter activities of ABCB1 and ABCC1. Furthermore, Twist1 silencing reverses the EMT phenotype, enhances the chemosensitivity of HCT-8/ V cells to anticancer agents in vitro and in vivo, and downregulates the expression of ABCB1 and ABCC1. Twist1-mediated promotion of ABCB1 and ABCC1 expression levels plays an important role in the drug resistance of colon cancer cells.

Reversal of P-gp-mediated multidrug resistance in colon cancer by cinobufagin.

Cinobufagin (CBF) is isolated from the skin and posterior auricular glands of the Asiatic toad (Bufo gargarizans). This study investigated the reversal effect of CBF on P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) in colon cancer. The effect of CBF on the cytotoxicity of anticancer drugs in P-gp overexpressing LoVo/ADR, HCT116/L, Cao-2/ADR cells and their parental cells was determined using CCK-8 assay. Apoptosis of anti-cancer drugs and accumulation of doxorubicin (DOX) and Rhodamine 123 (Rho123) in P-gp overexpressing cells were evaluated by flow cytometry. Results indicated that CBF significantly enhanced the sensitivity of P-gp substrate drugs on P-gp overexpressing cells, but had no effect on their parental cells. CBF enhanced the effect of DOX against P-gp-overexpressing LoVo/ADR cell xenografts in nude mice. Moreover, CBF also increased cell apoptosis of chemotherapy agents and intracellular accumulation of DOX and Rho123 in the MDR cells. Further research on the mechanisms revealed non-competitive inhibition of P-gp ATPase activity, but without altering the expression of P-gp. These findings demonstrated that CBF could be further developed into a safe and potent P-gp modulator for combination use with anticancer drugs in cancer chemotherapy.