Intracorporeal versus extracorporeal anastomosis in laparoscopic right hemicolectomy for overweight colon cancer patients: a case-control study.

INTRODUCTION: Either extracorporeal anastomosis (EA) or intracorporeal anastomosis (IA) could be selected for digestive reconstruction in laparoscopic right hemicolectomy (LRH). However, whether LRH with IA is feasible and beneficial for overweight right-side colon cancer (RCC) is unclear. This study aims to investigate the feasibility and advantage of IA in LRH for overweight RCC. METHODS: Forty-eight consecutive overweight RCC patients undergoing LRH with IA were matched with 48 consecutive cases undergoing LRH with EA. Both clinical and surgical data were collected and analyzed. RESULTS: The incidence of postoperative complications was 20.8% (10/48) in the EA group and 14.6% (7/48) in the IA group respectively, with no statistical difference. Compared to the EA group, patients in the IA group revealed faster gas (40.2 + 7.8 h vs. 45.6 + 7.9 h, P = 0.001) and stool discharge (4.0 + 1.2 d vs. 4.5 + 1.1 d, P = 0.040), shorter assisted incision (5.3 + 1.3 cm vs. 7.5 + 1.2 cm, P = 0.000), and less analgesic used (3.3 + 1.3 d vs. 4.0 + 1.3 d, P = 0.012). There were no significant differences in operation time, blood loss, or postoperative hospital stays. In the IA group, the first one third of cases presented longer operation time (228.4 + 29.3 min) compared to the middle (191.0 + 35.0 min, P = 0.003) and the last one third of patients (182.2 + 20.7 min, P = 0.000). CONCLUSION: LRH with IA is feasible and safe for overweight RCC, with faster bowel function recovery and less pain. Accumulation of certain cases of LRH with IA will facilitate surgical procedures and reduce operation time.

Nanodrug modified with engineered cell membrane targets CDKs to activate aPD-L1 immunotherapy against liver metastasis of immune-desert colon cancer.

Immunotherapy based on the PD-1/PD-L1 axis blockade has no benefit for patients diagnosed with colon cancer liver metastasis (CCLM) for the microsatellite stable/proficient mismatch repair (MSS/pMMR)) subtype, which is known as an immune-desert cancer featuring poor immunogenicity and insufficient CD8+ T cell infiltration in the tumor microenvironment. Here, a multifunctional nanodrug carrying a cyclin-dependent kinase (CDK)1/2/5/9 inhibitor and PD-L1 antibody is prepared to boost the immune checkpoint blockade (ICB)-based immunotherapy against MSS/pMMR CCLM via reversing the immunosuppressive tumor microenvironment. To enhance the MSS/pMMR CCLM-targeting efficacy, we modify the nanodrug with PD-L1 knockout cell membrane of this colon cancer subtype. First, CDKs inhibitor delivered by nanodrug down-regulates phosphorylated retinoblastoma and phosphorylated RNA polymerase II and meanwhile arrests the G2/M cell cycle in CCLM to promote immunogenic signal release, stimulate dendritic cell maturation, and enhance CD8+ T cell infiltration. Moreover, CDKi suppresses the secretion of immunosuppressive cytokines in tumor-associated myeloid cells sensitizing ICB therapy in CCLM. Notably, the great efficacy to activate immune responses is demonstrated in the patient-derived xenograft model and the patient-derived organoid model as well, revealing a clinical application potential. Overall, our study represents a promising therapeutic approach for targeting liver metastasis, remolding the tumor immune microenvironment (TIME), and enhancing the response of MSS/pMMR CCLM to boost ICB immunotherapy.

Erratum: CM082 Enhances the Efficacy of Chemotherapeutic Drugs by Inhibiting the Drug Efflux Function of ABCG2.

[This corrects the article DOI: 10.1016/j.omto.2019.12.007.].

Preservation versus resection of Denonvilliers' fascia in total mesorectal excision for male rectal cancer: follow-up analysis of the randomized PUF-01 trial.

Traditional total mesorectal excision (TME) for rectal cancer requires partial resection of Denonvilliers' fascia (DVF), which leads to injury of pelvic autonomic nerve and postoperative urogenital dysfunction. It is still unclear whether entire preservation of DVF has better urogenital function and comparable oncological outcomes. We conducted a randomized clinical trial to investigate the superiority of DVF preservation over resection (NCT02435758). A total of 262 eligible male patients were randomized to Laparoscopic TME with DVF preservation (L-DVF-P group) or resection procedures (L-DVF-R group), 242 of which completed the study, including 122 cases of L-DVF-P and 120 cases of L-DVF-R. The initial analysis of the primary outcomes of urogenital function has previously been reported. Here, the updated analysis and secondary outcomes including 3-year survival (OS), 3-year disease-free survival (DFS), and recurrence rate between the two groups are reported for the modified intention-to-treat analysis, revealing no significant difference. In conclusion, L-DVF-P reveals better postoperative urogenital function and comparable oncological outcomes for male rectal cancer patients.

New staging systems for left-sided colon cancer based on the number of retrieved and metastatic lymph nodes provide a more accurate prognosis.

Objectives: We aimed to explore reasonable lymph node classification strategies for left-sided colon cancer (LCC) patients. Methods: 48,425 LCC patients from 2010 to 2015 were identified in the US Surveillance, Epidemiology, and End Results database. We proposed an innovative revised nodal (rN) staging of the 8th American Joint Committee on Cancer (AJCC) Tumor/Node/Metastasis (TNM) classification based on the cut-off value of retrieved lymph nodes and survival analyses in patients with LCC. Log odds of positive lymph nodes (LODDS) stage is a numerical classification strategy obtained by a formula that incorporates the numbers of retrieved and positive lymph nodes. To develop the TrN or TLODDS classification, patients with similar survival rates were grouped by combining T and rN or LODDS stage. The TrN or TLODDS classification was further evaluated in a validation set of 12,436 LCC patients from 2016 to 2017 in the same database and a Chinese application set of 958 LCC patients. Results: We developed novel TrN and TLODDS classifications for LCC patients that incorporated 7 stages with reference to the AJCC staging system. In comparison to the 8th AJCC TNM and TrN classifications, TLODDS classification demonstrated significantly better discrimination (area under the receiver operating characteristic curve, 0.650 vs. 0.656 vs. 0.661, p < 0.001), better model-fitting (Akaike information criteria, 309,287 vs. 308,767 vs. 308,467), and superior net benefits. The predictive performance of the TrN and TLODDS classifications was further verified in the validation and application sets. Conclusion: Both the TrN and TLODDS classifications have better discriminatory ability, model-fitting, and net benefits than the existing TNM classification, and represent an alternative to the current TNM classification for LCC patients.

Identifying the E2F3-MEX3A-KLF4 signaling axis that sustains cancer cells in undifferentiated and proliferative state.

Rationale: Dysregulation of signaling that governs self-renewal and differentiation of intestinal stem cells (ISCs) is a major cause of colorectal cancer (CRC) initiation and progression. Methods: qRT-PCR, western blotting, in situ hybridization, immunohistochemistry and immunofluorescence assays were used to detect the expression levels of MEX3A, KLF4 and E2F3 in CRC tissues. The biological functions of MEX3A were studied using Mex3a knockout (KO) and intestinal epithelium specific conditional knockout (cKO) mice, AOM-DSS mouse colorectal tumor model, Apc floxed mouse tumor model and intestinal and tumor organoids. Transcriptomic RNA sequencing (RNA-seq), RNA crosslinking immunoprecipitation (CLIP) and luciferase reporter assays were performed to explore the molecular mechanisms of MEX3A. Results: RNA-binding protein MEX3A, a specific ISC marker gene, becomes ectopically upregulated upon CRC and its levels negatively correlate with patient survival prognosis. MEX3A functions as an oncoprotein that retains cancer cells in undifferentiated and proliferative status and it enhances their radioresistance to DNA damage. Mechanistically, a rate limiting factor of cellular proliferation E2F3 induces MEX3A, which in turn activates WNT pathway by directly suppressing expression of its pro-differentiation transcription factor KLF4. Knockdown of MEX3A with siRNA or addition of KLF4 agonist significantly suppressed tumor growth both by increasing differentiation status of cancer cells and by suppressing their proliferation. Conclusions: It identifies E2F3-MEX3A-KLF4 axis as an essential coordinator of cancer stem cell self-renewal and differentiation, representing a potent new druggable target for cancer differentiation therapy.

Synchronous Colorectal Liver Metastases considering Infectious Complications: Simultaneous or Delayed Surgery?

Background: Simultaneous or delayed surgery for synchronous colorectal liver metastases is performed in the clinic; which method is better is still up for debate. In particular, infectious complications are rarely compared. This study aims to investigate the differences between simultaneous and delayed surgery for synchronous colorectal liver metastases by comparing infectious complications and prognosis. Methods: Firstly, the patients' information from a single institution's database was retrospectively analyzed. Then the patients were divided into a simultaneous group and a delayed group according to synchronous colorectal liver metastases. Analyzing the postoperative complications within 30 days, the progression-free survival, and the overall survival in the two groups. Results: The simultaneous group had a higher neo-adjuvant chemotherapy rate (42.0% VS. 16.0% in the delayed group, P < 0.05) and laparoscopic surgery rate (89.8% VS. 72.0% in the delayed group, P < 0.05) than the delayed group. Moreover, the simultaneous group had a higher liver-related infection rate (17.0 VS. 0.0% in the delayed group, P < 0.05). Conclusion: Although there was no difference in survival rate between delayed and simultaneous surgeries, the delayed surgery have fewer liver-related infections compared with the simultaneous surgery in synchronous colorectal liver metastases patients. Delayed surgery could be a better treatment method for synchronous colorectal liver metastases patients.

Tumor-associated macrophage-specific CD155 contributes to M2-phenotype transition, immunosuppression, and tumor progression in colorectal cancer.

BACKGROUND: Onco-immunogenic molecule CD155 is overexpressed in various tumor microenvironments (TME) including in colorectal cancer (CRC). Tumor-associated macrophages (TAMs) are the most abundant immune cells in CRC TME and play a vital role in CRC progression and metastasis. Most studies have focused on investigating the role of CRC cell-specific CD155 on CRC progression, while the contribution of TAMs-specific CD155 is still unknown. Here, we sought to investigate the expression pattern of CD155 in CRC TAMs and its role in tumor immunity and progression. METHODS: CD155 expression patterns in CRC TAMs and macrophages in paratumor or adjacent normal tissue were analyzed in 50 patients with CRC using flow cytometry and in 141 patients with CRC using immunohistochemistry. The correlation of CD155 expression level in TAMs with M1 and M2 phenotypic transition was analyzed. The role of macrophage-specific CD155 in CRC progression and tumor immune response was investigated in vitro and in vivo. We further analyzed the effect of CRC cells on the regulation of CD155 expression in macrophages. RESULTS: CRC TAMs from clinical samples showed robustly higher expression of CD155 than macrophages from paratumor and adjacent normal tissues. The CD155 expression level was higher in TAMs of CRC at III/IV stages compared with the I/II stages and was negatively associated with the survival of patients with CRC. CD155+ TAMs showed an M2 phenotype and higher expression of interleukin (IL)-10 and transforming growth factor (TGF)-ß. CD155+ macrophages promoted CRC cell migration, invasion, and tumor growth supporting the findings from the clinical tissue analysis. This effect was mainly regulated by TGF-ß-induced STAT3 activation-mediated release of matrix metalloproteinases (MMP)2 and MMP9 in CRC cells. CD155-/- bone marrow transplantation in wild-type mice, as well as CD155- macrophages treatment, promoted the antitumor immune response in the mice ectopic CRC model. Additionally, CRC cells released IL-4 to trigger CD155 expression in macrophages indicating the regulatory role of CRC cells in the development of CD155+ TAMs. CONCLUSIONS: These findings indicated that CD155+ TAMs are responsible for the M2-phenotype transition, immunosuppression, and tumor progression in CRC. The specific localization of CD155+ TAMs in CRC tissue could turn into a potential therapeutic target for CRC treatment.

Case Report: Cetuximab in Combination With Chemotherapy for the Treatment of Multifocal Hepatic Metastases From Colorectal Cancer Guided by Genetic Tests.

Hepatic metastases were reported in up to 70% of colorectal cancer patients, among which multifocal hepatic metastasis represents one of the complications that lead to poor prognosis. The majority of the patients carrying multifocal hepatic metastases required pharmaceutical treatments to reduce the tumor size prior to surgical resection. However, the clinical responses to pharmaceutical agents were difficult to predict due to the heterogeneous nature of the multifocal tumors. Here, we report a case with multifocal hepatic metastases from colorectal cancer that was resistant to the primary chemotherapy and Bevacizumab plus chemotherapy, but responded to the combined therapy of Cetuximab and FOLFOX. Genetic tests had revealed that the tumor was highly metastatic due to the mutations of the WNT signaling pathway, and the metastatic tumors might be sensitive to Cetuximab. Consistent with the molecular characterizations, the metastatic tumors continue to emerge after chemotherapy, and rapidly relapsed in great numbers after liver resection. However, the combined therapy of Cetuximab and FOLFOX guided by the genetic tests significantly reduced the size and number of metastatic tumors. To conclude, deciphering the mutation profiles of multifocal metastatic tumors may guide the determination of treatment tactics, which may benefit the patients with non-resectable advanced carcinoma.

TIGIT promotes CD8+T cells exhaustion and predicts poor prognosis of colorectal cancer.

TIGIT is a lymphocyte surface receptor, which is mainly expressed on the surface of CD8+T cells. The role of TIGIT in colorectal cancer and its expression pattern in colorectal cancer infiltrating lymphocytes are still controversial. This study aimed at identifying the function of TIGIT in colorectal cancer. Patients with colorectal cancer showed significantly higher TIGIT+CD8+T cell infiltration in tumor tissues, metastases compared with paired PBMC and normal tissues through flow cytometry. TIGIT+CD8+T cells showed an exhausted phenotype and expressed low levels of killer cytokines IFN-γ, IL-2, TNF-α. In addition, more inhibitory receptors such as PD-1, LAG-3, and TIM-3 were expressed on the surface of TIGIT+CD8+T cells. TGF-ß1 could promote the expression of TIGIT and inhibit CD8+T cell function in vitro. Moreover, the accumulation of TIGIT+T cells in tumors was associated with advanced disease, predicted early recurrence, and reduced survival rates in colorectal cancer patients. Our results indicate that TIGIT can be a biological marker for the prognosis of colorectal cancer, and TIGIT can be used as a potential target for treatment.

Π electron-stabilized polymeric micelles potentiate docetaxel therapy in advanced-stage gastrointestinal cancer.

Gastrointestinal (GI) cancers are among the most lethal malignancies. The treatment of advanced-stage GI cancer involves standard chemotherapeutic drugs, such as docetaxel, as well as targeted therapeutics and immunomodulatory agents, all of which are only moderately effective. We here show that Π electron-stabilized polymeric micelles based on PEG-b-p(HPMAm-Bz) can be loaded highly efficiently with docetaxel (loading capacity up to 23 wt%) and potentiate chemotherapy responses in multiple advanced-stage GI cancer mouse models. Complete cures and full tumor regression were achieved upon intravenously administering micellar docetaxel in subcutaneous gastric cancer cell line-derived xenografts (CDX), as well as in CDX models with intraperitoneal and lung metastases. Nanoformulated docetaxel also outperformed conventional docetaxel in a patient-derived xenograft (PDX) model, doubling the extent of tumor growth inhibition. Furthermore, micellar docetaxel modulated the tumor immune microenvironment in CDX and PDX tumors, increasing the ratio between M1-and M2-like macrophages, and toxicologically, it was found to be very well-tolerated. These findings demonstrate that Π electron-stabilized polymeric micelles loaded with docetaxel hold significant potential for the treatment of advanced-stage GI cancers.

Effect of Denonvilliers' Fascia Preservation Versus Resection During Laparoscopic Total Mesorectal Excision on Postoperative Urogenital Function of Male Rectal Cancer Patients: Initial Results of Chinese PUF-01 Randomized Clinical Trial.

OBJECTIVE: To compare the outcomes of laparoscopic total mesorectal excision (L-TME) with Denonvilliers' fascia (DVF) preservation versus resection on urogenital function of male patients with rectal cancer. BACKGROUND: The protective effect of DVF during L-TME on pelvic autonomic nerves and postoperative urogenital function remains controversial. METHODS: Between August 26, 2015 and July 18, 2019, 253 male patients with cT1-4 (T1-2 for anterior wall) N0-2M0 rectal cancer from 11 institutions were enrolled, and randomly assigned to L-TME with DVF preservation (Exp-group, n = 123) or resection procedures (Con-group, n = 130). Urinary function was assessed by residual urine volume, maximal flow rate, and International Prostate Symptom Score; sexual function was assessed by 5-item version of the International Index of Erectile Function (IIEF-5) and ejaculation grading. RESULTS: The Exp-group patients showed a lower urinary dysfunction rate (6.8% vs 25.4%, P = 0.003), higher maximal flow rate (16.25 ±â€Š8.02 vs 12.40 ±â€Š7.05 mL/s, P = 0.007), and lower International Prostate Symptom Score (6.55 ±â€Š5.86 vs 8.57 ±â€Š5.85, P = 0.026) than the Con-group patients at 2 weeks after surgery. The incidence of erectile dysfunction (IIEF-5 ≤ 11) at 12 months after surgery was lower in the Exp-group than in the Con-group (12.5% vs 34.2%, P = 0.023); Exp-group manifested superior IIEF-5 (16.63 ±â€Š6.28 vs 12.26 ±â€Š6.83, P = 0.018). The incidence of ejaculation dysfunction was lower in the Exp-group than in the Con-group at 12 months after surgery (10.0% vs 29.4%, P = 0.034). CONCLUSIONS: DVF preservation during L-TME revealed protective effects on postoperative urogenital function, and could be a better choice for male rectal cancer patients with specific staging and location. TRIAL REGISTRATION NUMBER: NCT02435758.

SPT6 recruits SND1 to co-activate human telomerase reverse transcriptase to promote colon cancer progression.

Human telomerase reverse transcriptase (hTERT) plays an extremely important role in cancer initiation and development, including colorectal cancer (CRC). However, the precise upstream regulatory mechanisms of hTERT in different cancer types remain poorly understood. Here, we uncovered the candidate transcriptional factor of hTERT in CRC and explored its role and the corresponding molecular mechanisms in regulating hTERT expression and CRC survival with an aim of developing mechanism-based combinational targeting therapy. The possible binding proteins at the hTERT promoter were uncovered using pull-down/mass spectrometry analysis. The regulation of SPT6 on hTERT expression and CRC survival was evaluated in human CRC cell lines and mouse models. Mechanistic studies focusing on the synergy between SPT6 and staphylococcal nuclease and Tudor domain containing 1 (SND1) in controlling hTERT expression and CRC progression were conducted also in the above two levels. The expression correlation and clinical significance of SPT6, SND1, and hTERT were investigated in tumor tissues from murine models and patients with CRC in situ. SPT6 was identified as a possible transcriptional factor to bind to the hTERT promoter. SPT6 knockdown decreased the activity of hTERT promoter, downregulated the protein expression level of hTERT, suppressed proliferation, invasion, and stem-like properties, promoted apoptosis induction, and enhanced chemotherapeutic drug sensitivity in vitro. SPT6 silencing also led to the delay of tumor growth and metastasis in mice carrying xenografts of human-derived colon cancer cells. Mechanistically, SND1 interacted with SPT6 to co-control hTERT expression and CRC cell proliferation, stemness, and growth in vitro and in vivo. SPT6, SND1, and hTERT were highly expressed simultaneously in CRC tissues, both from the murine model and patients with CRC in situ, and pairwise expression among these three factors showed a significant positive correlation. In brief, our research demonstrated that SPT6 synergized with SND1 to promote CRC development by targeting hTERT and put forward that inhibiting the SPT6-SND1-hTERT axis may create a therapeutic vulnerability in CRC.

Targeting the NCOA3-SP1-TERT axis for tumor growth in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has a high mortality rate and lacks an effective therapeutic target. Elevated expression of human telomerase reverse transcriptase (TERT) is an important hallmark in cancers, but the mechanism by which TERT is activated differentially in cancers is poorly understood. Here, we have identified nuclear receptor coactivator-3 (NCOA3) as a new modulator of TERT expression and tumor growth in HCC. NACO3 specifically binds to the TERT promoter at the -234 to -144 region and transcriptionally activates TERT expression. NCOA3 promotes HCC cell growth and tumor progression in vitro and in vivo through upregulating the TERT signaling. Knockdown of NACO3 suppresses HCC cell viability and colony formation, whereas TERT overexpression rescues this suppression. NCOA3 interacts with and recruits SP1 binding on the TERT promoter. Knockdown of NCOA3 also inhibits the expression of the Wnt signaling-related genes but has no effect on the Notch signaling-targeting genes. Moreover, NCOA3 is positively correlated with TERT expression in HCC tumor tissues, and high expression of both NCOA3 and TERT predicts a poor prognosis in HCC patients. Our findings indicate that targeting the NCOA3-SP1-TERT signaling axis may benefit HCC patients.

Rociletinib (CO-1686) enhanced the efficacy of chemotherapeutic agents in ABCG2-overexpressing cancer cells in vitro and in vivo.

Overexpression of adenosine triphosphate (ATP)-binding cassette subfamily G member 2 (ABCG2) in cancer cells is known to cause multidrug resistance (MDR), which severely limits the clinical efficacy of chemotherapy. Currently, there is no FDA-approved MDR modulator for clinical use. In this study, rociletinib (CO-1686), a mutant-selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), was found to significantly improve the efficacy of ABCG2 substrate chemotherapeutic agents in the transporter-overexpressing cancer cells in vitro and in MDR tumor xenografts in nude mice, without incurring additional toxicity. Mechanistic studies revealed that in ABCG2-overexpressing cancer cells, rociletinib inhibited ABCG2-mediated drug efflux and increased intracellular accumulation of ABCG2 probe substrates. Moreover, rociletinib, inhibited the ATPase activity, and competed with [125I] iodoarylazidoprazosin (IAAP) photolabeling of ABCG2. However, ABCG2 expression at mRNA and protein levels was not altered in the ABCG2-overexpressing cells after treatment with rociletinib. In addition, rociletinib did not inhibit EGFR downstream signaling and phosphorylation of protein kinase B (AKT) and extracellular signal-regulated kinase (ERK). Our results collectively showed that rociletinib reversed ABCG2-mediated MDR by inhibiting ABCG2 efflux function, thus increasing the cellular accumulation of the transporter substrate anticancer drugs. The findings advocated the combination use of rociletinib and other chemotherapeutic drugs in cancer patients with ABCG2-overexpressing MDR tumors.

Investigation of the role and mechanism of ARHGAP5-mediated colorectal cancer metastasis.

Background: Metastatic colorectal cancer (CRC) is a lethal disease; however, the underlying molecular mechanisms remain unclear and require further study. Methods: RNA-Seq, PCR, Western blotting, immunohistochemistry, ChIP and RNAi assays were performed to investigate Rho GTPase-activating protein 5 (ARHGAP5, aslo known as p190RhoGAP-B, p190-B) expression and the clinical relevance, functional roles and regulatory mechanisms of this protein using human CRC cells and tissues. In vivo, two cell-based xenograft models were used to evaluate the roles of ARHGAP5 in CRC metastasis. Results: Here, we report that ARHGAP5 expression is significantly increased in metastatic CRC tissues and is inversely associated with patient overall survival. The suppression of ARHGAP5 reduces CRC cell metastasis in vitro and in cell-based xenograft models. Furthermore, we show that ARHGAP5 promotes CRC cell epithelial-mesenchymal transition by negatively regulating RhoA activity. Mechanistically, cAMP response element-binding protein (CREB1) transcriptionally upregulates ARHGAP5 expression, and decreased miR-137 further contributes to ARHGAP5 mRNA stability in CRC. Conclusions: Overall, our study highlights the crucial function of ARHGAP5 in CRC metastasis, thus suggesting novel prognostic biomarkers and hypothetical therapeutic targets.

Reversal of ABCB1-related multidrug resistance by ERK5-IN-1.

BACKGROUND: Inhibition of ABC transporters is considered the most effective way to circumvent multidrug resistance (MDR). In the present study, we evaluated the MDR modulatory potential of ERK5-IN-1, a potent extracelluar signal regulated kinase 5 (ERK5) inhibitor. METHODS: The cytotoxicity and MDR reversal effect of ERK5-IN-1 were assessed by MTT assay. The KBv200-inoculated nude mice xenograft model was used for the in vivo study. Doxorubicin efflux and accumulation were measured by flow cytometry. The modulation of ABCB1 activity was measured by colorimetric ATPase assay and [125I]-iodoarylazidoprazosin (IAAP) photolabeling assay. Effect of ERK5-IN-1 on expression of ABCB1 and its downstream markers was measured by PCR and/or Western blot. Cell surface expression and subcellular localization of ABCB1 were tested by flow cytometry and immunofluorescence. RESULTS: Our results showed that ERK5-IN-1 significantly increased the sensitivity of vincristine, paclitaxel and doxorubicin in KBv200, MCF7/adr and HEK293/ABCB1 cells, respectively. This effect was not found in respective drug sensitive parental cell lines. Moreover, in vivo combination studies showed that ERK5-IN-1 effectively enhanced the antitumor activity of paclitaxel in KBv200 xenografts without causing addition toxicity. Mechanistically, ERK5-IN-1 increased intracellular accumulation of doxorubicin dose dependently by directly inhibiting the efflux function of ABCB1. ERK5-IN-1 stimulated the ABCB1 ATPase activity and inhibited the incorporation of [125I]-iodoarylazidoprazosin (IAAP) into ABCB1 in a concentration-dependent manner. In addition, ERK5-IN-1 treatment neither altered the expression level of ABCB1 nor blocked the phosphorylation of downstream Akt or Erk1/2. No significant reversal effect was observed on ABCG2-, ABCC1-, MRP7- and LRP-mediated drug resistance. CONCLUSIONS: Collectively, these results indicated that ERK5-IN-1 efficiently reversed ABCB1-mediated MDR by competitively inhibiting the ABCB1 drug efflux function. The use of ERK5-IN-1 to restore sensitivity to chemotherapy or to prevent resistance could be a potential treatment strategy for cancer patients.

CM082 Enhances the Efficacy of Chemotherapeutic Drugs by Inhibiting the Drug Efflux Function of ABCG2.

The overexpression of ATP-binding cassette (ABC) transporters is one of the important mechanisms of multidrug resistance (MDR). Some tyrosine kinase inhibitors (TKIs) such as CM082 might be a potential ABC transporter inhibitor, thus potentially reversing MDR. We used a 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide (MTT) assay to determine the cytotoxicity and reversal effect of CM082. A xenograft model was established to evaluate the reversal MDR efficacy in vivo. The intracellular accumulation and efflux of ABCG2 substrates were measured by flow cytometry. We investigated the binding sites of ABCG2 via photolabeling ABCG2 with [125I]-iodoarylazidoprazosin (IAAP). Quantitative real-time PCR and western blot were utilized to analyze mRNA and protein expression. We found that CM082 could enhance the efficacy of substrate in ABCG2-overexpressing cells both in vitro and in vivo. Furthermore, CM082 significantly increased intracellular accumulation of ABCG2 substrates by inhibiting the efflux activity. CM082 stimulated ABCG2 ATPase activity and competed with [125I]-IAAP photolabeling of ABCG2 in a concentration-dependent manner. However, CM082 did not alter ABCG2 expression at protein and mRNA levels or inhibit vascular endothelial growth factor (VEGF) downstream signaling of AKT and extracellular signal-regulated kinase (ERK). Further research is encouraged to confirm whether CM082 concomitant with anticancer drugs of ABCG2 substrates could improve the clinical outcomes of cancer treatment in cancer patients with ABCG2 overexpression.

Aspirin enhances the sensitivity of colon cancer cells to cisplatin by abrogating the binding of NF-κB to the COX-2 promoter.

Cisplatin is one of the most potent chemotherapeutic agents for the treatment of colon cancer. Nevertheless, the unavoidability of the notable toxicity and the development of the acquired resistance severely restricted its clinical application. Aspirin and some other non-steroidal anti-inflammatory drugs have been used to prevent colon tumorigenesis as chemopreventive agents. Here, we explored the possibility of aspirin as an adjuvant drug to boost the anti-cancer effect of cisplatin for colon cancer. We found that aspirin significantly enhanced the cisplatin-mediated inhibitions of cell proliferation, migration and invasion and the induction of apoptosis in colon cancer cells. The combined treatment of aspirin and cisplatin suppressed the expression of the anti-apoptotic protein Bcl-2 and the EMT-related proteins, up-regulated the levels of the cleaved PARP and Bax, and blocked the PI3K/AKT and RAF-MEK-ERK signaling pathway. In addition, we demonstrated that the enhanced effect of aspirin on the cisplatin-induced inhibition of tumor cell growth was also mediated through the suppression of the binding activity of NF-κB to the COX-2 promoter. The combination of aspirin and cisplatin effectively attenuated the translocation of NF-κB p65/p50 from the cytoplasm to the nucleus, and abrogated the binding of NF-κB p65/p50 to the COX-2 promoter, thereby down-regulating COX-2 expression and PGE2 synthesis. Moreover, the in vivo study also verified the enhanced anti-tumor activity of such combined therapy in colon cancer by targeting the NF-κB/COX-2 signaling. Our results provided new insights into understanding the molecular mechanisms of aspirin in sensitizing cisplatin-mediated chemotherapeutic effect in colon cancer and indicated a great potential of this combined therapy for cancer treatment.

Endothelial progenitor cells promote viability and nerve regenerative ability of mesenchymal stem cells through PDGF-BB/PDGFR-ß signaling.

Denervation-induced erectile dysfunction (ED) is a prevailing health problem. Our previous study revealed that endothelial progenitor cells (EPCs) promoted the effect of mesenchymal stem cells (MSCs) on restoration of denervation-induced ED in rats. However, underling mechanisms are still largely elusive. In this study, EPCs and MSCs were co-cultured and resorted to co-EPCs and co-MSCs. EPCs-derived paracrine factors containing PDGF-BB (platelet-derived growth factor) were detected, and MSCs were pre-treated with PDGF-BB, while co-MSCs were pre-treated with PDGFR inhibitor AG1296. Either viability or nerve regenerative ability of MSCs was evaluated. In addition, inhibition of either PI3K/Akt or MEK/Erk pathway was performed to evaluate the role of PI3K/Akt and MEK/Erk pathway in PDGF-BB-induced viability of MSCs. The results revealed that PDGF-BB significantly increased the proportion of PDGFR-ß+ MSCs, and promoted both in-vitro and in-vivo viability, as well as nerve regenerative capacity and erectile function restoration of MSCs in rats. Inhibition of PI3K/Akt, MEK/Erk pathway or mTOR led to decrease of PDGF-BB/PDGFR-ß induced viability of MSCs. To our knowledge, our study first demonstrates that EPCs promote viability and potential nerve regenerative ability of MSCs through PDGF-BB/PDGFR-ß signaling and its downstream PI3K/Akt and MEK/Erk pathways. mTOR acts as a co-mediator in PI3K/Akt and MEK/Erk pathways.