Prognostic value of preoperative carcinoembryonic antigen/tumor size in rectal cancer.

BACKGROUND: Carcinoembryonic antigen (CEA) is a commonly used biomarker in colorectal cancer. However, controversy exists regarding the insufficient prognostic value of preoperative serum CEA alone in rectal cancer. Here, we combined preoperative serum CEA and the maximum tumor diameter to correct the CEA level, which may better reflect the malignancy of rectal cancer. AIM: To assess the prognostic impact of preoperative CEA/tumor size in rectal cancer. METHODS: We retrospectively reviewed 696 stage I to III rectal cancer patients who underwent curative tumor resection from 2007 to 2012. These patients were randomly divided into two cohorts for cross-validation: training cohort and validation cohort. The training cohort was used to generate an optimal cutoff point and the validation cohort was used to further validate the model. Maximally selected rank statistics were used to identify the optimum cutoff for CEA/tumor size. The Kaplan-Meier method and log-rank test were used to plot the survival curve and to compare the survival data. Univariate and multivariate Cox regression analyses were used to determine the prognostic value of CEA/tumor size. The primary and secondary outcomes were overall survival (OS) and disease-free survival (DFS), respectively. RESULTS: In all, 556 patients who satisfied both the inclusion and exclusion criteria were included and randomly divided into the training cohort (2/3 of 556, n = 371) and the validation cohort (1/3 of 556, n = 185). The cutoff was 2.429 ng/mL per cm. Comparison of the baseline data showed that high CEA/tumor size was correlated with older age, high TNM stage, the presence of perineural invasion, high CEA, and high carbohydrate antigen 19-9 (CA 19-9). Kaplan-Meier curves showed a manifest reduction in 5-year OS (training cohort: 56.7% vs 81.1%, P < 0.001; validation cohort: 58.8% vs 85.6%, P < 0.001) and DFS (training cohort: 52.5% vs 71.9%, P = 0.02; validation cohort: 50.3% vs 79.3%, P = 0.002) in the high CEA/tumor size group compared with the low CEA/tumor size group. Univariate and multivariate analyses identified CEA/tumor size as an independent prognostic factor for OS (training cohort: hazard ratio (HR) = 2.18, 95% confidence interval (CI): 1.28-3.73, P = 0.004; validation cohort: HR = 4.83, 95%CI: 2.21-10.52, P < 0.001) as well as DFS (training cohort: HR = 1.47, 95%CI: 0.93-2.33, P = 0.096; validation cohort: HR = 2.61, 95%CI: 1.38-4.95, P = 0.003). CONCLUSION: Preoperative CEA/tumor size is an independent prognostic factor for patients with stage I-III rectal cancer. Higher CEA/tumor size is associated with worse OS and DFS.

Bioprocessed Production of Resveratrol-Enriched Rice Wine: Simultaneous Rice Wine Fermentation, Extraction, and Transformation of Piceid to Resveratrol from Polygonum cuspidatum Roots.

A new bioprocess to produce resveratrol-enriched rice wine was established and the effects of adding Polygonum cuspidatum root powder to rice wine fermentation were investigated. In this new process, piceid and resveratrol were extracted from P. cuspidatum roots to rice wine and piceid was converted to resveratrol by ß-glucosidase during fermentation. After 10 days co-fermentation, rice wine with high levels of resveratrol was obtained, which contained ~14% (v/v) ethanol, 122 mg/L piceid, and 86 mg/L resveratrol. The resveratrol-enriched rice wine had enhanced antioxidant activity with significantly stronger 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, ferric ion reducing power, and ferrous ion chelating capability. Ultrafiltration (UF) was employed in this study using hollow fibers to clarify the end product, increase shelf life without heat treatment, and maintain the quality of the phenolic compounds. The boiled and UF-treated rice wine were evaluated for ethanol, piceid, resveratrol, clarity, aerobic plate count, total acidity, pH, reducing sugars, and amino acids. The quality of the resveratrol-enriched rice wine was maintained after four weeks storage at normal refrigeration temperatures.

Comparison of Artificial Neural Networks and Response Surface Methodology towards an Efficient Ultrasound-Assisted Extraction of Chlorogenic Acid from Lonicera japonica.

Chlorogenic acid (CGA), a bioactive compound commonly found in plants, has been demonstrated possessing nutraceutical potential in recent years. However, the more critical issue concerning how to improve production efficacy of CGA is still limited. It is a challenge to harvest a large amount of CGA without prolonging extraction time. In this study, the feasibility of using ultrasound for CGA extraction from Lonicera japonica was investigated. A central composite design (CCD) was employed to evaluate the effects of the operation parameters, including temperature, ethanol concentration, liquid to solid ratio, and ultrasound power on CGA yields. Meanwhile, the process of ultrasound-assisted extraction was optimized through modeling response surface methodology (RSM) and artificial neural network (ANN). The data indicated that CGA was efficiently extracted from the flower of Lonicera japonica by ultrasound assistance. The optimal conditions for the maximum extraction of CGA were as follows: The temperature at 33.56 °C, ethanol concentration at 65.88%, L/S ratio at 46:1 mL/g and ultrasound power at 150 W. ANN possessed greater optimization capacity than RSM for fitting experimental data and predicting the extraction process to obtain a maximum CGA yield. In conclusion, the process of ultrasound-assisted extraction can be well established by a methodological approach using either RSM or ANN, but it is worth mentioning that the ANN model used here showed the superiority over RSM for predicting and optimizing.

Nomograms for predicting pathological response to neoadjuvant treatments in patients with rectal cancer.

BACKGROUND: In recent decades, neoadjuvant therapy (NT) has been the standardized treatment for locally advanced rectal cancer (LARC). Approximately 8%-35% of patients with LARC who received NT were reported to have achieved a complete pathological response (pCR). If the pathological response (PR) can be accurately predicted, these patients may not need surgery. In addition, no response after NT implies that the tumor is destructive, resistant to both chemotherapy and radiotherapy, and prone to having a high metastatic potential. Therefore, developing accurate models to predict PR has great clinical significance and can help achieve individualized treatment in LARC patients. AIM: To establish nomograms for predicting PR to different NT regimens based on pretreatment parameters for patients with LARC. METHODS: Rectal cancer patients were identified from the database of The Sixth Affiliated Hospital, Sun Yat-sen University from January 2012 to December 2016. Logistic regression and nomograms were developed to predict the probability of pCR and good downstaging to ypT0-2N0M0 (ypTNM 0-I), respectively, based on pretreatment parameters for all LARC patients. Nomograms were also developed for three NT regimens (capecitabine/deGramont-RT, mFOLFOX6, and mFOLFOX6-RT) to predict pCR probability. RESULTS: Four hundred and three patients were included in this study; 72 (17.9%) had pCR at the final pathology report, and 177 (43.9%) achieved good downstaging to ypT0-2N0M0 (ypTNM 0-I). The nomogram for predicting pCR probability showed that NT regimens, tumor differentiation, mesorectal fascia (MRF) status, and tumor length significantly influenced pCR probability. When predicting the probability of good downstaging, tumor differentiation, MRF status, and clinical T stage were the significant factors. Nomograms were developed based on NT regimens. For the capecitabine/de Gramont-RT group, the multivariate analysis showed that the neutrophil-lymphocyte ratio (NLR) was the only significant factor, thus we could not develop a nomogram for this regimen. For the mFOLFOX6-RT group, the analysis showed that the significant factors were tumor length and MRF status; and for the mFOLFOX6 group, the significant factors were tumor length and tumor differentiation. CONCLUSION: We established accurate nomograms for predicting the PR to preoperative NT regimens based on pretreatment parameters for LARC patients.

Optimization of ultrasonic-assisted enzymatic hydrolysis conditions for the production of antioxidant hydrolysates from porcine liver by using response surface methodology.

OBJECTIVE: The objective of this study was to optimize ultrasonic-assisted enzymatic hydrolysis conditions, including enzyme-to-substrate (E/S) ratio, pH, and temperature, for producing porcine liver hydrolysates (PLHs) with the highest 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity by using response surface methodology (RSM). METHODS: The study used RSM to determine the combination of hydrolysis parameters that maximized the antioxidant activity of our PLHs. Temperature (40°C, 54°C, and 68°C), pH (8.5, 9.5, and 10.5), and E/S ratio (0.1%, 2.1%, and 4.1%) were selected as the independent variables and analyzed according to the preliminary experiment results, whereas DPPH free radical scavenging activity was selected as the dependent variable. RESULTS: Analysis of variance showed that E/S ratio, pH, and temperature significantly affected the hydrolysis process (p<0.01). The optimal conditions for producing PLHs with the highest scavenging activity were as follows: E/S ratio, 1.4% (v/w); temperature, 55.5°C; and initial pH, 10.15. Under these conditions, the degree of hydrolysis, DPPH free radical scavenging activity, ferrous ion chelating ability, and reducing power of PLHs were 24.12%, 79%, 98.18%, and 0.601 absorbance unit, respectively. The molecular weight of most PLHs produced under these optimal conditions was less than 5,400 Da and contained 45.7% hydrophobic amino acids. CONCLUSION: Ultrasonic-assisted enzymatic hydrolysis can be applied to obtain favorable antioxidant hydrolysates from porcine liver with potential applications in food products for preventing lipid oxidation.

Antioxidant properties of porcine liver proteins hydrolyzed using Monascus purpureus.

In this study, the antioxidant activities of porcine liver proteins, hydrolyzed using Alcalase®, papain, pepsin, or a microbial suspension of Monascus purpureus (APLH, PaPLH, PePLH, and MPLH, respectively), were investigated. The results indicated that the yield and degree of hydrolysis (DH) of hydrolysates increased with hydrolysis time. The highest yield and peptide content were obtained from APLH, whereas the DH of PaPLH was higher than that of the others. MPLH exhibited the highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and reducing power, whereas APLH and PaPLH exhibited the higher ferrous ion-chelating ability than that of the MPLH. The molecular weights of all the hydrolysates were <10 kDa. The PaPLH exhibited the highest contents of total amino acids and hydrophobic amino acids. Fifteen antioxidant fractions obtained from MPLH contained one or more of the following amino acids in their sequences: Tyr, Trp, Ala, Pro, Met, Lys, Asp, Cys, Val, Leu, and His.

Association of perioperative blood pressure with long-term survival in rectal cancer patients.

BACKGROUND: Several studies suggested that hypertension is positively related to cancer incidence and mortality. In this study, we investigated the association between perioperative blood pressure (BP) and long-term survival outcomes in patients with rectal cancer. METHODS: This study included a cohort of 358 patients with stages I-III rectal cancer who underwent a curative resection between June 2007 and June 2011. Both pre- and postoperative BPs were measured, by which patients were grouped (low BP: <120/80 mmHg; high BP: ≥120/80 mmHg). The survival outcomes were compared between these two groups. The primary endpoints were disease-free survival (DFS) and cancer-specific survival (CSS). RESULTS: Univariate analysis showed that patients with high preoperative systolic BP had lower 3-year DFS (67.2% vs. 82.1%, P = 0.041) and CSS rates (81.9% vs. 94.8%, P = 0.003) than patients with low preoperative systolic BP, and the associations remained significant in the Cox multivariate analysis, with the adjusted hazard ratios equal to 1.97 [95% confidence interval (CI) = 1.08-3.60, P = 0.028] and 2.85 (95% CI = 1.00-8.25, P = 0.050), respectively. Similarly, in postoperative evaluation, patients with high systolic BP had significantly lower 3-year CSS rates than those with low systolic BP (78.3% vs. 88.9%, P = 0.032) in univariate analysis. Moreover, high pre- and/or postoperative systolic BP presented as risk factors for CSS in the subgroups of patients who did not have a history of hypertension, with and/or without perioperative administration of antihypertensive drugs. CONCLUSIONS: High preoperative systolic BP was an independent risk factor for both CSS and DFS rates, and high postoperative systolic BP was significantly associated with a low CSS rate in rectal cancer patients. Additionally, our results suggest that rectal cancer patients may get survival benefit from BP control in perioperative care. However, further studies should be conducted to determine the association between BP and CSS and targets of BP control.

SET antagonist enhances the chemosensitivity of non-small cell lung cancer cells by reactivating protein phosphatase 2A.

SET is known as a potent PP2A inhibitor, however, its oncogenic role including its tumorigenic potential and involvement in the development of chemoresistance in non-small cell lung cancer (NSCLC) has not yet been fully discussed. In present study, we investigated the oncogenic role of SET by SET-knockdown and showed that SET silencing impaired cell growth rate, colony formation and tumor sphere formation in A549 cells. Notably, silencing SET enhanced the pro-apoptotic effects of paclitaxel, while ectopic expression of SET diminished the sensitivity of NSCLC cells to paclitaxel. Since the SET protein was shown to affect chemosensitivity, we next examined whether combining a novel SET antagonist, EMQA, sensitized NSCLC cells to paclitaxel. Both the in vitro and in vivo experiments suggested that EMQA and paclitaxel combination treatment was synergistic. Importantly, we found that downregulating p-Akt by inhibiting SET-mediated protein phosphatase 2A (PP2A) inactivation determined the pro-apoptotic effects of EMQA and paclitaxel combination treatment. To dissect the critical site for EMQA functioning, we generated several truncated SET proteins. By analysis of the effects of EMQA on the binding affinities of different truncated SET proteins to PP2A-catalytic subunits, we revealed that the 227-277 amino-acid sequence is critical for EMQA-induced SET inhibition. Our findings demonstrate the critical role of SET in NSCLC, particularly in the development of chemoresistance. The synergistic effects of paclitaxel and the SET antagonist shown in current study encourage further validation of the clinical potential of this combination.

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an independent prognostic marker in wild-type KRAS metastatic colorectal cancer after colorectal liver metastasectomy.

BACKGROUND: The impact of KRAS signaling on cancerous inhibitor of protein phosphatase 2A (CIP2A) expression has not yet been explored. We investigated the impact of KRAS on CIP2A expression in colorectal cancer patients after colorectal liver metastasectomy. METHODS: We examined CIP2A expression by immunohistochemistry (IHC) and used direct sequencing to identify the mutational status of KRAS exon 2 (codon 12 and 13). The association between CIP2A expression, KRAS genotype, clinicopathological parameters and survival were examined by the Kaplan-Meier method and the Cox proportional hazards model. A combination of immunoblotting and proliferation assays were employed to elucidate the role of CIP2A in signal transduction pathways in wild-type KRAS Caco-2 cells. RESULTS: A total of 220 colorectal cancer patients who had undergone colorectal liver metastasectomy were included in the study. The mutant KRAS genotype was associated with CIP2A overexpression. CIP2A expression was an independent prognostic marker in patients with wild-type KRAS metastatic colorectal cancer after colorectal liver metastasectomy (relative risk = 1.873, P = 0.019). Targeted silencing of CIP2A in Caco-2 cells (wild-type KRAS) led to decreased expression of pERK/ERK and decreased cell proliferation. Overexpression of mutant KRAS G12D in Caco-2 cells led to an increase in CIP2A expression and cell proliferation. In Caco-2 cells with the KRAS G12D, KRAS overexpression preserved the regulation effect of CIP2A in KRAS and abrogated the impact of CIP2A regulation on pERK/ERK and cell proliferation. CIP2A inhibition also increased the efficacy of cetuximab in Caco-2 cells. CONCLUSIONS: CIP2A is an independent prognostic marker in patients with wild-type KRAS metastatic colorectal cancer after colorectal liver metastasectomy.

STAT3 mediates regorafenib-induced apoptosis in hepatocellular carcinoma.

PURPOSE: Here, we aim to investigate the molecular mechanism of regorafenib and verify the potential druggable target for the treatment of hepatocellular carcinoma (HCC). EXPERIMENTAL DESIGN: HCC cell lines (PLC5, HepG2, Hep3B, SK-Hep1, and HA59T) were used to investigate the in vitro effect of regorafenib. Phosphatase activity was analyzed in HCC cells and purified SHP-1 proteins. PLC5-bearing mice were used to test the therapeutic efficiency of 20 and 40 mg/kg/d treatment with regorafenib ([Formula: see text] mice). The clinical relevance of STAT3 signaling was investigated with 142 tumor samples from different patients with HCC. Descriptive statistical analysis was used to compare the baseline characteristics of patients and the expression of p-STAT3. RESULTS: Regorafenib inhibited STAT3-related signaling in a dose-dependent manner and was a more potent inhibitor of STAT3 than sorafenib. Regorafenib increased SHP-1 phosphatase activity in purified SHP-1 protein directly. N-SH2 domain deletion and D61A mutants mimicking open-form SHP-1 partially abolished regorafenib-induced STAT3 inhibition and apoptosis. Importantly, a higher level of expression of STAT3 was found in patients with advanced clinical stages (P = 0.009) and poorly differentiated tumors (P = 0.035). CONCLUSIONS: Regorafenib induced significant tumor inhibition by relieving the autoinhibited N-SH2 domain of SHP-1 directly and inhibiting p-STAT3 signals. STAT3 may be suitable as a prognostic marker of HCC development, and may be a druggable target for HCC-targeted therapy using regorafenib.

SHP-1 is a target of regorafenib in colorectal cancer.

Regorafenib is an inhibitor of multiple protein kinases which exerts antitumor and antimetastatic activities in metastatic colorectal cancer (CRC). SH2 domain-containing phosphatase 1 (SHP-1) is reported to have tumor suppressive potential because it acts as a negative regulator of p-STAT3(Tyr705) signaling. However, little is known about the mechanism regarding regorafenib affects SHP-1 tyrosine phosphatase activity and leads to apoptosis and tumor suppression in CRC. Here, we found that regorafenib triggered apoptotic cell death and significantly enhanced SHP-1 activity, which dramatically decreased the phosphorylated form of STAT3 at Tyr705 (p-STAT3(Tyr705)). Importantly, regorafenib augmented SHP-1 activity by direct disruption of the association between N-SH2 and catalytic PTP domain of SHP-1. Deletion of the N-SH2 domain (dN1) or point mutation (D61A) of SHP-1 blocked the effect of regorafenib-induced SHP-1 activity, growth inhibition and a decrease of p-STAT3(Tyr705) expression, suggesting that regorafenib triggers a conformational change in SHP-1 by relieving its autoinhibition. In vivo assay showed that regorafenib significantly inhibited xenograft growth and decreased p-STAT3(Tyr705) expression but induced higher SHP-1 activity. Collectively, regorafenib is a novel SHP-1 agonist exerts superior anti-tumor effects by enhancing SHP-1 activity that directly targets p-STAT3(Tyr705). Small molecule-enhancement of SHP-1 activity may be a promising therapeutic approach for CRC treatment.

Nintedanib (BIBF-1120) inhibits hepatocellular carcinoma growth independent of angiokinase activity.

BACKGROUND & AIMS: Nintedanib, a triple angiokinase inhibitor, is currently being evaluated against advanced HCC in phase I/II clinical trials. Here, we report the underlying molecular mechanism by which nintedanib (BIBF-1120) induces an anti-HCC effect. METHODS: To further elucidate whether the effect of nintedanib on SHP-1 is dependent on its angiokinase inhibition activity, we developed a novel kinase-independent derivative of nintedanib, ΔN. HCC cell lines were treated with nintedanib or its derivative (ΔN) and apoptosis, signal transduction, and phosphatase activity were analyzed. Purified SHP-1 proteins or HCC cells expressing deletion N-SH2 domain or D61A point mutants were used to investigate the potential effect of nintedanib on SHP-1. In vivo efficacy was determined in nude mice with HCC subcutaneous xenografts (n⩾8 mice). RESULTS: Nintedanib induced anti-proliferation in HCC cell lines by targeting STAT3. Ectopic STAT3 abolished nintedanib-mediated apoptosis in HCC cells. Nintedanib further activated SHP-1 in purified SHP-1 proteins suggesting that nintedanib directly affects SHP-1 for STAT3 inhibition. HCC cells or recombinant SHP-1 proteins expressing deletion of N-SH2 domain or D61A mutants restored the activity of nintedanib suggesting that the auto-inhibition structure of SHP-1 was relieved by nintedanib. Although ΔN only retained the backbone of nintedanib without kinase activity, ΔN still induced substantial anti-HCC activity in vitro and in vivo by targeting STAT3. CONCLUSIONS: Nintedanib induced significant anti-HCC activity independent of angiokinase inhibition activity in a preclinical HCC model by relieving autoinhibition of SHP-1. Our findings provide new mechanistic insight into the inhibition of HCC growth by nintedanib.

Comparison of short- and long-term outcomes after extralevator abdominoperineal excision and standard abdominoperineal excision for rectal cancer: a systematic review and meta-analysis.

PURPOSE: Whether the introduction of extralevator abdominoperineal excision (ELAPE) improves survival and safety remains controversial. We conducted a systematic review and meta-analysis of all comparative studies to define the efficacy and safety of ELAPE and standard abdominoperineal excision (APE). MATERIALS AND METHODS: A search for all major databases and relevant journals from inception to July 2013 without restriction on languages or regions was performed. Outcome measures were the oncological parameters of circumferential resection margin (CRM) involvement, intraoperative bowel perforation (IOP), and local recurrence, as well as other parameters of blood loss, operative time, length of hospitalization, and postoperative complication. The test of heterogeneity was performed with the Q statistic. RESULTS: A total of 949 patients were included in the meta-analysis. Oncological pooled estimates of intraoperative bowel perforation rate (RR 0.34; 95 % CI 0.21-0.54; P < 0.00001), CRM involvement (RR 0.44; 95 % CI 0.34-0.56; P < 0.00001), and local recurrence (RR 0.32; 95 % CI 0.14-0.74; P = 0.008) all showed outcomes that were significantly lower in ELAPE than in APE. A similar incidence of postoperative complication was attributed to both groups, including overall complication (RR 0.93; 95 % CI 0.66-1.32; P = 0.69), perineal wound complication (RR 0.72; 95 % CI 0.33-1.55; P = 0.39), and urinary dysfunction (RR 1.53; 95 % CI 0.88-2.67; P = 0.13). CONCLUSION: ELAPE has a lower intraoperative bowel perforation rate, positive CRM rate, and local recurrence rate than APE. There is evidence that in selected low rectal cancer patients, ELAPE is a more efficient and equally safe option to replace APE. Due to the inherent limitations of the present study, future randomized controlled trials will be useful to confirm this conclusion.

Bortezomib congeners induce apoptosis of hepatocellular carcinoma via CIP2A inhibition.

CIP2A is an oncoprotein that upregulates p-Akt and promotes cancer cell proliferation and survival. The proteasome inhibitor bortezomib has been shown to reduce CIP2A and lead to cell apoptosis. Here; we modified the functional group of bortezomib to generate a series of novel compounds and conducted a structure-activity relationship (SAR) study. The results showed that compound 1 was able to repress CIP2A expression and cell apoptosis in the same manner as bortezomib, but with less potency in inhibition of proteasome activity. This finding provides a new direction for the design of CIP2A inhibitors.

Nilotinib induces autophagy in hepatocellular carcinoma through AMPK activation.

Hepatocellular carcinoma (HCC) is the most common liver cancer and the third-leading cause of cancer death worldwide. Nilotinib is an orally available receptor tyrosine kinase inhibitor approved for chronic myelogenous leukemia. This study investigated the effect of nilotinib on HCC. Nilotinib did not induce cellular apoptosis. Instead, staining with acridine orange and microtubule-associated protein 1 light chain 3 revealed that nilotinib induced autophagy in a dose- and time-dependent manner in HCC cell lines, including PLC5, Huh-7, and Hep3B. Moreover, nilotinib up-regulated the phosphryaltion of AMP-activated kinase (AMPK) and protein phosphatase PP2A inactivation were detected after nilotinib treatment. Up-regulating PP2A activity suppressed nilotinib-induced AMPK phosphorylation and autophagy, suggesting that PP2A mediates the effect of nilotinib on AMPK phosphorylation and autophagy. Our data indicate that nilotinib-induced AMPK activation is mediated by PP2A, and AMPK activation and subsequent autophagy might be a major mechanism of action of nilotinib. Growth of PLC5 tumor xenografts in BALB/c nude mice was inhibited by daily oral treatment with nilotinib. Western blot analysis showed both increased phospho-AMPK expression and decreased PP2A activity in vivo. Together, our results reveal that nilotinib induces autophagy, but not apoptosis in HCC, and that the autophagy-inducing activity is associated with PP2A-regulated AMPK phosphorylation.

Cancerous inhibitor of protein phosphatase 2A mediates bortezomib-induced autophagy in hepatocellular carcinoma independent of proteasome.

Previously, we reported that cancerous inhibitor of protein phosphatase 2A (CIP2A) mediates the apoptotic effect of bortezomib in hepatocellular carcinoma (HCC). Here, we report a proteasome-independent mechanism by which bortezomib induces autophagy in HCC. Our data indicate that bortezomib activated autophagy in a dose- and time- dependent manner in HCC cell lines including Huh-7, Sk-Hep1, and Hep3B. Bortezomib downregulated CIP2A, phospho-Akt (P-Akt) and phospho-4EBP1 (P-4EBP1) in a dose- and time-dependent manner in all tested HCC cells. Ectopic expression of CIP2A abolished the effect of bortezomib on autophagy. Co-treatment of bortezomib and calyculin A, a PP2A inhibitor, reduced the effect of bortezomib on P-Akt, P-4EBP1, and autophagy. Increased phosphorylation of either Akt or 4EBP1 by ectopic overexpression protected cells from bortezomib-induced autophagy. Furthermore, we examined the effect of ΔBtz, a bortezomib derivative that closely resembles bortezomib structurally but has no proteasome activity, in HCC. Interestingly, ΔBtz demonstrated similar effects to bortezomib on autophagy, CIP2A, P-Akt and P-4EBP1, suggesting that the effect of bortezomib on autophagy is independent of proteasome inhibition. Moreover, our in vivo data showed that both bortezomib and ΔBtz inhibited tumor growth, downregulated CIP2A, P-Akt and induced autophagy in Huh-7 tumors. In conclusion, bortezomib induces autophagy in HCC through a CIP2A-PP2A-Akt-4EBP1 pathway.

Inhibition of CIP2A determines erlotinib-induced apoptosis in hepatocellular carcinoma.

Erlotinib is a small-molecular inhibitor of epidermal growth factor receptor (EGFR). Here, we identify that cancerous inhibitor of protein phosphatase 2A (CIP2A) is a major determinant mediating erlotinib-induced apoptosis in hepatocellular carcinoma (HCC). Erlotinib showed differential effects on apoptosis in 4 human HCC cell lines. Erlotinib induced significant apoptosis in Hep3B and PLC5 cell lines; however, Huh-7 and HA59T cell lines showed resistance to erlotinib-induced apoptosis at all tested doses. Down-regulation of CIP2A, a cellular inhibitor of protein phosphatase 2A (PP2A), mediated the apoptotic effect of erlotinib in HCC. Erlotinib inhibited CIP2A in a dose- and time-dependent manner in all sensitive HCC cells whereas no alterations in CIP2A were found in resistant cells. Overexpression of CIP2A upregulated phospho-Akt and protected Hep3B cells from erlotinib-induced apoptosis. In addition, silencing CIP2A by siRNA restored the effects of erlotinib in Huh-7 cells. Moreover, adding okadaic acid, a PP2A inhibitor, abolished the effects of erlotinib on apoptosis in Hep3B cells; and forskolin, a PP2A agonist enhanced the effect of erlotinib in resistant HA59T cells. Combining Akt inhibitor MK-2206 with erlotinib restored the sensitivity of HA59T cells to erlotinib. Furthermore, in vivo xenograft data showed that erlotinib inhibited the growth of PLC5 tumor but had no effect on Huh-7 tumor. Erlotinib downregulated CIP2A and upregulated PP2A activity in PLC5 tumors, but not in Huh-7 tumors. In conclusion, inhibition of CIP2A determines the effects of erlotinib on apoptosis in HCC. CIP2A may be useful as a therapeutic biomarker for predicting clinical response to erlotinib in HCC treatment.

Inhibition of Bcl-2 improves effect of LCL161, a SMAC mimetic, in hepatocellular carcinoma cells.

In this study, we investigated the effect of LCL161, a SMAC mimetic, in hepatocellular carcinoma (HCC). LCL161 showed differential effects on apoptosis in four HCC cell lines, and the endogenous level of Bcl-2 determined the sensitivity of HCC cells to LCL161. Cytotoxicity and apoptosis were observed in sensitive PLC5 and Hep3B cells that express lower levels of Bcl-2, but not in resistant Huh-7 and SK-Hep1 cells with higher Bcl-2 expression. Down regulation of Bcl-2 by small interference RNA overcame the resistance to LCL161 in Huh-7, and the apoptotic effect was rescued in Bcl-2-expressing Hep3B. To test the hypothesis that Bcl-2 determines the sensitivity of HCC cells to LCL161, we assayed the biological effect of SC-2001, a novel Bcl-2 inhibitor derived from obatoclax, in LCL161-resistant cell lines. Huh-7 cells co-treated with LCL161 and SC-2001 showed a significant dose-dependent apoptotic effect demonstrated by sub-G1 assay and cleavage of PARP. Furthermore, the combination index (CI) of LCL161 and SC-2001 showed a convincing synergism in resistant Huh-7. In addition, the combinational therapy showed significant growth inhibition in Huh-7-bearing xenograft tumors. Notably, down regulation of Bcl-2 was observed in a tumor sample treated with LCL161 and SC-2001. In conclusion, targeting Bcl-2 with SC-2001 overcomes drug resistance to LCL161 in HCC cells thus suggesting a new anti-IAP combinational therapy for HCC.

Bortezomib sensitizes HCC cells to CS-1008, an antihuman death receptor 5 antibody, through the inhibition of CIP2A.

Previously, we have shown that bortezomib overcame TRAIL resistance in hepatocellular carcinoma (HCC) cells via the inhibition of Akt. Here, we report that bortezomib sensitizes these TRAIL-resistant cells, including Huh-7, Hep3B, and Sk-Hep1, to CS-1008, a humanized agonistic antihuman death receptor 5 antibody. Cancerous inhibitor of protein phosphatase 2A (CIP2A) mediated the sensitizing effect of bortezomib to CS-1008 through inhibiting protein phosphatase 2A (PP2A) activity. Combination treatment of bortezomib and CS-1008 downregulated CIP2A in a concentration- and time-dependent manner, and increased PP2A activity in HCC cells. Importantly, ectopic expression of CIP2A decreased Akt-related PP2A activity, indicating that CIP2A negatively regulates Akt-related PP2A activity in HCC cells. Moreover, silencing CIP2A by short interfering RNA enhanced CS-1008-induced apoptosis in HCC cells and ectopic expression of CIP2A in HCC cells abolished CS-1008-induced apoptosis, indicating that CIP2A plays an important role in the sensitizing effect of bortezomib to CS-1008. Finally, our in vivo data showed that CS-1008 and bortezomib combination treatment decreased tumor growth significantly. In conclusion, bortezomib sensitized HCC cells to CS-1008 through the inhibition of CIP2A.

Synergistic interactions between sorafenib and bortezomib in hepatocellular carcinoma involve PP2A-dependent Akt inactivation.

BACKGROUND & AIMS: Previously we reported that Akt inactivation determines the sensitivity of hepatocellular carcinoma (HCC) cells to bortezomib. Here we report that combined treatment with sorafenib and bortezomib shows synergistic effects in HCC. METHODS: HCC cell lines (PLC/PRF/5, Huh-7, and Hep3B) were treated with sorafenib and/or bortezomib and analyzed in terms of apoptosis signal transduction. In vivo efficacy was determined in nude mice with PLC/PRF/5 xenografts. RESULTS: Pretreatment with sorafenib enhanced bortezomib-induced apoptotic cell death by restoring bortezomib's ability to inactivate Akt in PLC/PRF/5 cells. Knocking down Akt1 by RNA-interference overcame apoptotic resistance to bortezomib in PLC/PRF/5 cells and ectopic expression of active Akt in HCC cells abolished the bortezomib sensitizing effect of sorafenib, indicating Akt inactivation plays a key role in mediating the combinational effects. Moreover, okadaic acid, a protein phosphatase 2A (PP2A) inhibitor, reversed down-regulation of phospho-Akt (P-Akt) expression induced by co-treatment with sorafenib and bortezomib, and 1, 9 di-deoxy-forskolin, a PP2A agonist, restored bortezomib's effect on P-Akt and apoptosis. Importantly, silencing of PP2A by RNA-interference reduced the apoptotic effect induced by sorafenib-bortezomib co-treatment, indicating that PP2A is indispensable for mediating the effects of these drugs. Notably, sorafenib with bortezomib increased PP2A activity in PLC/PRF/5 cells without altering protein levels of PP2A complex or the interaction between PP2A and Akt proteins. Finally, sorafenib plus bortezomib significantly suppressed PLC/PRF/5 xenograft tumor growth, down-regulated P-Akt expression, and up-regulated PP2A activity. CONCLUSIONS: The combination of sorafenib and bortezomib shows synergy in HCC through PP2A-dependent Akt inactivation.