Blockade of growth hormone receptor signaling by using pegvisomant: A functional therapeutic strategy in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is an aggressive neoplasm with poor clinical outcome because most patients present at an advanced stage, at which point curative surgical options, such as tumor excision or liver transplantation, are not feasible. Therefore, the majority of HCC patients require systemic therapy. Nonetheless, the currently approved systemic therapies have limited effects, particularly in patients with advanced and resistant disease. Hence, there is a critical need to identify new molecular targets and effective systemic therapies to improve HCC outcome. The liver is a major target of the growth hormone receptor (GHR) signaling, and accumulating evidence suggests that GHR signaling plays an important role in HCC pathogenesis. We tested the hypothesis that GHR could represent a potential therapeutic target in this aggressive neoplasm. We measured GH levels in 767 HCC patients and 200 healthy controls, and then carried out clinicopathological correlation analyses. Moreover, specific inhibition of GHR was performed in vitro using siRNA and pegvisomant (a small peptide that blocks GHR signaling and is currently approved by the FDA to treat acromegaly) and in vivo, also using pegvisomant. GH was significantly elevated in 49.5% of HCC patients, and these patients had a more aggressive disease and poorer clinical outcome (P<0.0001). Blockade of GHR signaling with siRNA or pegvisomant induced substantial inhibitory cellular effects in vitro. In addition, pegvisomant potentiated the effects of sorafenib (P<0.01) and overcame sorafenib resistance (P<0.0001) in vivo. Mechanistically, pegvisomant decreased the phosphorylation of GHR downstream survival proteins including JAK2, STAT3, STAT5, IRS-1, AKT, ERK, and IGF-IR. In two patients with advanced-stage HCC and high GH who developed sorafenib resistance, pegvisomant caused tumor stability. Our data show that GHR signaling represents a novel "druggable" target, and pegvisomant may function as an effective systemic therapy in HCC. Our findings could also lead to testing GHR inhibition in other aggressive cancers.

Disruption of Growth Hormone Receptor Signaling Abrogates Hepatocellular Carcinoma Development.

Introduction: Hepatocellular carcinoma (HCC) is the most common type of primary liver cancers. It is an aggressive neoplasm with dismal outcome because most of the patients present with an advanced-stage disease, which precludes curative surgical options. Therefore, these patients require systemic therapies that typically induce small improvements in overall survival. Hence, it is crucial to identify new and promising therapeutic targets for HCC to improve the current outcome. The liver is a key organ in the signaling cascade triggered by the growth hormone receptor (GHR). Previous studies have shown that GHR signaling stimulates the proliferation and regeneration of liver cells and tissues; however, a definitive role of GHR signaling in HCC pathogenesis has not been identified. Methods: In this study, we used a direct and specific approach to analyze the role of GHR in HCC development. This approach encompasses mice with global (Ghr-/- ) or liver-specific (LiGhr-/- ) disruption of GHR expression, and the injection of diethylnitrosamine (DEN) to develop HCC in these mice. Results: Our data show that DEN induced HCC in a substantial majority of the Ghr+/+ (93.5%) and Ghr +/- (87.1%) mice but not in the Ghr-/- (5.6%) mice (P < 0.0001). Although 57.7% of LiGhr-/- mice developed HCC after injection of DEN, these mice had significantly fewer tumors than LiGhr+/+ (P < 0.001), which implies that the expression of GHR in the liver cells might increase tumor burden. Notably, the pathologic, histologic, and biochemical characteristics of DEN-induced HCC in mice resembled to a great extent human HCC, despite the fact that etiologically this model does not mimic this cancer in humans. Our data also show that the effects of DEN on mice livers were primarily related to its carcinogenic effects and ability to induce HCC, with minimal effects related to toxic effects. Conclusion: Collectively, our data support an important role of GHR in HCC development, and suggest that exploiting GHR signaling may represent a promising approach to treat HCC.

Enhancing Antigen Retrieval to Unmask Signaling Phosphoproteins in Formalin-fixed Archival Tissues.

The introduction of targeted therapy has revolutionized cancer treatment. Nonetheless, for this approach to succeed, it is crucial to identify the targets, particularly when activated, in tumor tissues. Phosphorylation is a posttranslational modification that causes activation of numerous oncogenic protein kinases and transcription regulators. Hence, phosphoproteins is a class of biomarkers that has therapeutic and prognostic implications directly relevant to cancer patients' management. Despite the progress in histopathology methodology, analysis of the expression of phosphoproteins in tumor tissues still represents a challenge owing to preanalytical and analytical factors that include antigen retrieval strategies. In this study, we tested the hypothesis that optimizing antigen retrieval methods will improve phosphoproteins unmasking and enhance their immunohistochemical staining signal. We screened 4 antigen retrieval methods by using antibodies specific for 3 oncogenic phosphoproteins to stain human lymphoma tumors that were developed in severe combined immunodeficiency mice and subsequently fixed in formalin for 2 years. Then, we used antibodies specific for 15 survival phosphoproteins to compare the most effective method identified from our screening experiment to the antigen retrieval method that is most commonly utilized. Using the antigen retrieval buffer Tris-EDTA at pH 9.0 and heating for 45 minutes at 97°C unmasked and significantly enhanced the staining of 9 of the 15 phosphoproteins (P<0.0001). Our antigen retrieval approach is cost effective and feasible for clinical and research settings. We anticipate that combining this approach with the newly proposed methods to improve tissue fixation will further improve unmasking of phosphoproteins in human and animal tissues.

Clinically Applicable Machine Learning Approaches to Identify Attributes of Chronic Kidney Disease (CKD) for Use in Low-Cost Diagnostic Screening.

OBJECTIVE: Chronic kidney disease (CKD) is a major public health concern worldwide. High costs of late-stage diagnosis and insufficient testing facilities can contribute to high morbidity and mortality rates in CKD patients, particularly in less developed countries. Thus, early diagnosis aided by vital parameter analytics using affordable computer-aided diagnosis could not only reduce diagnosis costs but improve patient management and outcomes. METHODS: In this study, we developed machine learning models using selective key pathological categories to identify clinical test attributes that will aid in accurate early diagnosis of CKD. Such an approach will save time and costs for diagnostic screening. We have also evaluated the performance of several classifiers with k-fold cross-validation on optimized datasets derived using these selected clinical test attributes. RESULTS: Our results suggest that the optimized datasets with important attributes perform well in diagnosis of CKD using our proposed machine learning models. Furthermore, we evaluated clinical test attributes based on urine and blood tests along with clinical parameters that have low costs of acquisition. The predictive models with the optimized and pathologically categorized attributes set yielded high levels of CKD diagnosis accuracy with random forest (RF) classifier being the best performing. CONCLUSIONS: Our machine learning approach has yielded effective predictive analytics for CKD screening which can be developed as a resource to facilitate improved CKD screening for enhanced and timely treatment plans.

Perspectives on natural compounds in chemoprevention and treatment of cancer: an update with new promising compounds.

Cancer is the second deadliest disease worldwide. Although recent advances applying precision treatments with targeted (molecular and immune) agents are promising, the histological and molecular heterogeneity of cancer cells and huge mutational burdens (intrinsic or acquired after therapy) leading to drug resistance and treatment failure are posing continuous challenges. These recent advances do not negate the need for alternative approaches such as chemoprevention, the pharmacological approach to reverse, suppress or prevent the initial phases of carcinogenesis or the progression of premalignant cells to invasive disease by using non-toxic agents. Although data are limited, the success of several clinical trials in preventing cancer in high-risk populations suggests that chemoprevention is a rational, appealing and viable strategy to prevent carcinogenesis. Particularly among higher-risk groups, the use of safe, non-toxic agents is the utmost consideration because these individuals have not yet developed invasive disease. Natural dietary compounds present in fruits, vegetables and spices are especially attractive for chemoprevention and treatment because of their easy availability, high margin of safety, relatively low cost and widespread human consumption. Hundreds of such compounds have been widely investigated for chemoprevention and treatment in the last few decades. Previously, we reviewed the most widely studied natural compounds and their molecular mechanisms, which were highly exploited by the cancer research community. In the time since our initial review, many promising new compounds have been identified. In this review, we critically review these promising new natural compounds, their molecular targets and mechanisms of anticancer activity that may create novel opportunities for further design and conduct of preclinical and clinical studies.

Insulin-like growth factor 1/Child-Turcotte-Pugh composite score as a predictor of treatment outcomes in patients with advanced hepatocellular carcinoma treated with sorafenib.

BACKGROUND: Sorafenib was the first systemic therapy approved for the treatment of Child-Turcotte-Pugh (CTP) class A patients with advanced hepatocellular carcinoma (HCC). However, there are no biomarkers to predict survival and treatment outcomes and guide HCC systemic therapy. Type 1 insulin-like growth factor (IGF-1)/CTP composite score has emerged as a potential hepatic reserve assessment tool. Our study investigated the association of the IGF/CTP score with overall survival (OS) and progression-free survival (PFS) of HCC patients treated with sorafenib. MATERIALS AND METHODS: In this prospective study, patients with HCC were treated with sorafenib and followed up until progression/death. We calculated the IGF/CTP score and used the Kaplan-Meier method and log-rank test to estimate and compare the time-to-event outcomes between patient subgroups. RESULTS: 171 patients were included, 116 of whom were CTP class A. Median PFS for IGF/CTP score AA and AB patients were 6.88 and 4.28 months, respectively (p = 0.1359). Median OS for IGF/CTP score AA and AB patients were 14.54 and 7.60 months, respectively (p = 0.1378). The PFS and OS was superior in AA patients, but the difference was not significant, likely due to the sample size. However, there was a significant difference in early OS and PFS curves between AA and AB (p = 0.0383 and p = 0.0099), respectively. CONCLUSIONS: In CTP class A patients, IGF/CTP score B was associated with shorter PFS and OS, however, study was underpowered to reach statistical significance. If validated in larger cohorts, IGF/CTP score may serve as stratification tool in clinical trials, a hepatic reserve assessment tool for HCC outcomes prediction and to assist in therapy decisions.

HepatoScore-14: Measures of Biological Heterogeneity Significantly Improve Prediction of Hepatocellular Carcinoma Risk.

BACKGROUND AND AIMS: Therapeutic, clinical trial entry and stratification decisions for hepatocellular carcinoma (HCC) are made based on prognostic assessments, using clinical staging systems based on small numbers of empirically selected variables that insufficiently account for differences in biological characteristics of individual patients' disease. APPROACH AND RESULTS: We propose an approach for constructing risk scores from circulating biomarkers that produce a global biological characterization of individual patient's disease. Plasma samples were collected prospectively from 767 patients with HCC and 200 controls, and 317 proteins were quantified in a Clinical Laboratory Improvement Amendments-certified biomarker testing laboratory. We constructed a circulating biomarker aberration score for each patient, a score between 0 and 1 that measures the degree of aberration of his or her biomarker panel relative to normal, which we call HepatoScore. We used log-rank tests to assess its ability to substratify patients within existing staging systems/prognostic factors. To enhance clinical application, we constructed a single-sample score, HepatoScore-14, which requires only a subset of 14 representative proteins encompassing the global biological effects. Patients with HCC were split into three distinct groups (low, medium, and high HepatoScore) with vastly different prognoses (medial overall survival 38.2/18.3/7.1 months; P < 0.0001). Furthermore, HepatoScore accurately substratified patients within levels of existing prognostic factors and staging systems (P < 0.0001 for nearly all), providing substantial and sometimes dramatic refinement of expected patient outcomes with strong therapeutic implications. These results were recapitulated by HepatoScore-14, rigorously validated in repeated training/test splits, concordant across Myriad RBM (Austin, TX) and enzyme-linked immunosorbent assay kits, and established as an independent prognostic factor. CONCLUSIONS: HepatoScore-14 augments existing HCC staging systems, dramatically refining patient prognostic assessments and therapeutic decision making and enrollment in clinical trials. The underlying strategy provides a global biological characterization of disease, and can be applied broadly to other disease settings and biological media.

Impact of Integrating Insulin-Like Growth Factor 1 Levels into Model for End-Stage Liver Disease Score for Survival Prediction in Hepatocellular Carcinoma Patients.

BACKGROUND: Liver reserve affects survival in hepatocellular carcinoma (HCC). Model for End-Stage Liver Disease (MELD) score is used to predict overall survival (OS) and to prioritize HCC patients on the transplantation waiting list, but more accurate models are needed. We hypothesized that integrating insulin-like growth factor 1 (IGF-1) levels into MELD score (MELD-IGF-1) improves OS prediction as compared to MELD. METHODS: We measured plasma IGF-1 levels in training (n = 310) and validation (n = 155) HCC cohorts and created MELD-IGF-1 score. Cox models were used to determine the association of MELD and MELD-IGF-1 with OS. Harrell's c-index was used to compare the predictive capacity. RESULTS: IGF-1 was significantly associated with OS in both cohorts. Patients with an IGF-1 level of ≤26 ng/mL in the training cohort and in the validation cohorts had significantly higher hazard ratios than patients with the same MELD but IGF-1 >26 ng/mL. In both cohorts, MELD-IGF-1 scores had higher c-indices (0.60 and 0.66) than MELD scores (0.58 and 0.60) (p < 0.001 in both cohorts). Overall, 26% of training and 52.9% of validation cohort patients were reclassified into different risk groups by MELD-IGF-1 (p < 0.001). CONCLUSIONS: After independent validation, the MELD-IGF-1 could be used to risk-stratify patients in clinical trials and for priority assignment for patients on liver transplantation waiting list.

Molecular Profiling of Hepatocellular Carcinoma Using Circulating Cell-Free DNA.

PURPOSE: Molecular profiling has been used to select patients for targeted therapy and determine prognosis. Noninvasive strategies are critical to hepatocellular carcinoma (HCC) given the challenge of obtaining liver tissue biopsies. EXPERIMENTAL DESIGN: We analyzed blood samples from 206 patients with HCC using comprehensive genomic testing (Guardant Health) of circulating tumor DNA (ctDNA). RESULTS: A total of 153/206 (74.3%) were men; median age, 62 years (range, 18-91 years). A total of 181/206 patients had ≥1 alteration. The total number of alterations was 680 (nonunique); median number of alterations/patient was three (range, 1-13); median mutant allele frequency (% cfDNA), 0.49% (range, 0.06%-55.03%). TP53 was the common altered gene [>120 alterations (non-unique)] followed by EGFR, MET, ARID1A, MYC, NF1, BRAF, and ERBB2 [20-38 alterations (nonunique)/gene]. Of the patients with alterations, 56.9% (103/181) had ≥1 actionable alterations, most commonly in MYC, EGFR, ERBB2, BRAF, CCNE1, MET, PIK3CA, ARID1A, CDK6, and KRAS. In these genes, amplifications occurred more frequently than mutations. Hepatitis B (HBV)-positive patients were more likely to have ERBB2 alterations, 35.7% (5/14) versus 8.8% HBV-negative (P = 0.04). CONCLUSIONS: This study represents the first large-scale analysis of blood-derived ctDNA in HCC in United States. The genomic distinction based on HCC risk factors and the high percentage of potentially actionable genomic alterations suggests potential clinical utility for this technology.

Dual inhibition of IGF-IR and ALK as an effective strategy to eradicate NPM-ALK+ T-cell lymphoma.

BACKGROUND: Nucleophosmin-anaplastic lymphoma kinase-expressing (NPM-ALK+) T cell lymphoma is an aggressive neoplasm. NPM-ALK, an oncogenic tyrosine kinase, plays a critical role in this lymphoma. Recently, selective ALK inhibitors have emerged as a first-line therapy for this neoplasm. Unfortunately, ALK inhibitors were hindered by emergence of resistance and relapse. We have previously demonstrated that type I insulin-like growth factor receptor (IGF-IR) is commonly expressed and activated in this lymphoma. In addition, IGF-IR and NPM-ALK are physically associated and reciprocally enhance their phosphorylation/activation. Herein, we tested the hypothesis that combined inhibition of IGF-IR and NPM-ALK could significantly improve the effects of inhibiting each kinase alone. METHODS: We used clinically utilized inhibitors of IGF-IR (picropodophyllin; PPP) and ALK (ASP3026) to assess the in vitro cellular effects of combined treatment versus treatment using a single agent. Moreover, we used a systemic NPM-ALK+ T cell lymphoma mouse model to analyze the in vivo effects of PPP and ASP3026 alone or in combination. RESULTS: Our data show that combined treatment with PPP and ASP3026 decreased the viability, proliferation, and anchorage-independent colony formation, and increased apoptosis of NPM-ALK+ T cell lymphoma cells in vitro. The in vitro effects of combined treatment were synergistic and significantly more pronounced than the effects of PPP or ASP3026 alone. Biochemically, simultaneous antagonism of IGF-IR and ALK induced more pronounced decrease in pIGF-IRY1135/1136, pNPM-ALKY646, and pSTAT3Y705 levels than antagonizing IGF-IR or ALK alone. Moreover, combined targeting of IGF-IR and NPM-ALK decreased significantly systemic lymphoma tumor growth and improved mice survival in vivo. Consistent with the in vitro results, the in vivo effects of the combined therapy were more pronounced than the effects of targeting IGF-IR or ALK alone. CONCLUSIONS: Combined targeting of IGF-IR and ALK is more effective than targeting IGF-IR or ALK alone in NPM-ALK+ T cell lymphoma. This strategy might also limit emergence of resistance to high doses of ALK inhibitors. Therefore, it could represent a successful therapeutic approach to eradicate this aggressive lymphoma. Importantly, combined inhibition is feasible because of the clinical availability of IGF-IR and ALK inhibitors. Our findings are applicable to other types of cancer where IGF-IR and ALK are simultaneously expressed.

Autotaxin determines colitis severity in mice and is secreted by B cells in the colon.

Autotaxin (ATX or ENPP2) is a secreted lysophospholipase D that produces lysophosphatidic acid (LPA), a pleiotropic lipid mediator acting on specific GPCRs. ATX and LPA have been implicated in key (patho)physiologic processes, including embryonic development, lymphocyte homing, inflammation, and cancer progression. Using LPA receptor knockout mice, we previously uncovered a role for LPA signaling in promoting colitis and colorectal cancer. Here, we examined the role of ATX in experimental colitis through inducible deletion of Enpp2 in adult mice. ATX expression was increased upon induction of colitis, whereas ATX deletion reduced the severity of inflammation in both acute and chronic colitis, accompanied by transient weight loss. ATX expression in lymphocytes was strongly reduced in Rag1-/- and µMT mice, suggesting B cells as a major ATX-producing source, which was validated by immunofluorescence and biochemical analyses. ATX secretion by B cells from control, but not Enpp2 knockout, mice led to ERK activation in colorectal cancer cells and promoted T cell migration. We conclude that ATX deletion suppresses experimental colitis and that B cells are a major source of ATX in the colon. Our study suggests that pharmacological inhibition of ATX could be a therapeutic strategy in colitis.-Lin, S., Haque, A., Raeman, R., Guo, L., He, P., Denning, T. L., El-Rayes, B., Moolenaar, W. H., Yun, C. C. Autotaxin determines colitis severity in mice and is secreted by B cells in the colon.

Inhibition of autotaxin alleviates inflammation and increases the expression of sodium-dependent glucose cotransporter 1 and Na+/H+ exchanger 3 in SAMP1/Fc mice.

Crohn's disease (CD) is a chronic, relapsing, inflammatory disease that is often associated with malnutrition because of inflammation in the small intestine. Autotaxin (ATX) is a secreted enzyme that produces extracellular lysophosphatidic acid. Increasing evidence suggests that ATX is upregulated during inflammation, and inhibition of ATX has been effective in attenuating chronic inflammatory conditions, such as arthritis and pulmonary fibrosis. This study aims to determine whether inhibition of ATX alleviates CD-associated inflammation and malnutrition by using SAMP1/Fc mice, a model of CD-like ileitis. SAMP1/Fc mice were treated the ATX inhibitor PF-8380 for 4 wk. Inhibition of ATX led to increased weight gain in SAMP1/Fc mice, decreased T helper 2 cytokine expression, including IL-4, IL-5, and IL-13, and attenuated immune cell migration. SAMP1/Fc mice have low expression of Na+-dependent glucose transporter 1 (SGLT1), suggesting impaired nutrient absorption associated with ileitis. PF-8380 treatment significantly enhanced SGLT1 expression in SAMP1/Fc mice, which could reflect the increased weight changes. However, IL-4 or IL-13 did not alter SGLT1 expression in Caco-2 cells, ruling out their direct effects on SGLT1 expression. Immunofluorescence analysis showed that the expression of sucrase-isomaltase, a marker for intestinal epithelial cell (IEC) differentiation, was decreased in inflamed regions of SAMP1/Fc mice, which was partially restored by PF-8380. Moreover, expression of Na+/H+ exchanger 3 was also improved by PF-8380, suggesting that suppression of inflammation by PF-8380 enhanced IEC differentiation. Our study therefore suggests that ATX is a potential target for treating intestinal inflammation and restoration of the absorptive function of the intestine. NEW & NOTEWORTHY This study is the first, to our knowledge, to determine whether autotoxin (ATX) inhibition improves inflammation and body weights in SAMP1/Fc mice, a mouse model of ileitis. ATX inhibition increased body weights of SAMP1/Fc mice and increased Na+-dependent glucose transporter 1 (SGLT1) expression. Increased SGLT1 expression in the inflamed regions was not a direct effect of cytokines but an indirect effect of increased epithelial cell differentiation upon ATX inhibition.

In Vitro and In Vivo Synergistic Antitumor Activity of the Combination of BKM120 and Erlotinib in Head and Neck Cancer: Mechanism of Apoptosis and Resistance.

We previously reported that the EGFR-targeted inhibitor erlotinib induces G1 arrest of squamous cell carcinoma of the head and neck (SCCHN) cell lines without inducing significant apoptosis. Large-scale genomic studies suggest that >50% of SCCHN cases have activation of PI3K pathways. This study investigated whether cotargeting of EGFR and PI3K has synergistic antitumor effects and apoptosis induction. We examined growth suppression, apoptosis, and signaling pathway modulation resulting from single and combined targeting of EGFR and PI3K with erlotinib and BKM120, respectively, in a panel of SCCHN cell lines and a xenograft model of SCCHN. In a panel of 12 cell lines, single targeting of EGFR with erlotinib or PI3K with BKM120 suppressed cellular growth without inducing significant apoptosis. Cotargeting of EGFR and PI3K synergistically inhibited SCCHN cell line and xenograft tumor growth, but induced variable apoptosis; some lines were highly sensitive, others were resistant. Mechanistic studies revealed that the combination inhibited both axes of the mTORC1 (S6 and 4EBP1) pathway in apoptosis-sensitive cell lines along with translational inhibition of Bcl-2, Bcl-xL, and Mcl-1, but failed to inhibit p-4EBP1, Bcl-2, Bcl-xL, and Mcl-1 in an apoptosis-resistant cell line. siRNA-mediated knockdown of eIF4E inhibited Bcl-2 and Mcl-1 and sensitized this cell line to apoptosis. Our results strongly suggest that cotargeting of EGFR and PI3K is synergistic and induces apoptosis of SCCHN cell lines by inhibiting both axes of the AKT-mTOR pathway and translational regulation of antiapoptotic Bcl-2 proteins. These findings may guide the development of clinical trials using this combination of agents. Mol Cancer Ther; 16(4); 729-38. ©2017 AACR.

Preclinical In Vitro, In Vivo, and Pharmacokinetic Evaluations of FLLL12 for the Prevention and Treatment of Head and Neck Cancers.

Despite its high promise for cancer prevention and therapy, the potential utility of curcumin in cancer is compromised by its low bioavailability and weak potency. The purpose of the current study was to assess the in vitro and in vivo efficacy and pharmacokinetic parameters of the potent curcumin analogue FLLL12 in SCCHN and identify the mechanisms of its antitumor effect. IC50 values against a panel of one premalignant and eight malignant head and neck cancer cell lines as well as apoptosis assay results suggested that FLLL12 is 10- to 24-fold more potent than natural curcumin depending on the cell line and induces mitochondria-mediated apoptosis. In vivo efficacy (xenograft) and pharmacokinetic studies also suggested that FLLL12 is significantly more potent and has more favorable pharmacokinetic properties than curcumin. FLLL12 strongly inhibited the expression of p-EGFR, EGFR, p-AKT, AKT, Bcl-2, and Bid and increased the expression of Bim. Overexpression of constitutively active AKT or Bcl-2 or ablation of Bim or Bid significantly inhibited FLLL12-induced apoptosis. Further mechanistic studies revealed that FLLL12 regulated EGFR and AKT at transcriptional levels, whereas Bcl-2 was regulated at the translational level. Finally, FLLL12 strongly inhibited the AKT downstream targets mTOR and FOXO1a and 3a. Taken together, our results strongly suggest that FLLL12 is a potent curcumin analogue with more favorable pharmacokinetic properties that induces apoptosis of head and neck cancer cell lines by inhibition of survival proteins including EGFR, AKT, and Bcl-2 and increasing of the proapoptotic protein Bim.

Combination of erlotinib and EGCG induces apoptosis of head and neck cancers through posttranscriptional regulation of Bim and Bcl-2.

Combinatorial approaches using two or more compounds are gaining increasing attention for cancer therapy. We have previously reported that the combination of the EGFR-TKI erlotinib and epigallocatechin-3-gallate (EGCG) exhibited synergistic chemopreventive effects in head and neck cancers by inducing the expression of Bim, p21, p27, and by inhibiting the phosphorylation of ERK and AKT and expression of Bcl-2. In the current study, we further investigated the mechanism of regulation of Bim, Bcl-2, p21 and p27, and their role in apoptosis. shRNA-mediated silencing of Bim significantly inhibited apoptosis induced by the combination of erlotinib and EGCG (p = 0.005). On the other hand, overexpression of Bcl-2 markedly protected cells from apoptosis (p = 0.003), whereas overexpression of constitutively active AKT only minimally protected cells from apoptosis induced by the combination of the two compounds. Analysis of mRNA expression by RT-PCR revealed that erlotinib, EGCG and their combination had no significant effects on the mRNA expression of Bim, p21, p27 or Bcl-2 suggesting the post-transcriptional regulation of these molecules. Furthermore, we found that erlotinib or the combination of EGCG and erlotinib inhibited the phosphorylation of Bim and stabilized Bim after inhibition of protein translation by cycloheximide. Taken together, our results strongly suggest that the combination of erlotinib and EGCG induces apoptosis of SCCHN cells by regulating Bim and Bcl-2 at the posttranscriptional level.

FLLL12 induces apoptosis in lung cancer cells through a p53/p73-independent but death receptor 5-dependent pathway.

Unlike chemotherapy drugs, the safety of natural compounds such as curcumin has been well established. However, the potential use of curcumin in cancer has been compromised by its low bioavailability, limited tissue distribution and rapid biotransformation leading to low in vivo efficacy. To circumvent these problems, more potent and bioavailable analogs have been synthesized. In the current study, we investigated the mechanism of anti-tumor effect of one such analog, FLLL12, in lung cancers. IC50 values measured by sulforhodamine B (SRB) assay at 72 h and apoptosis assays (annexin V staining, cleavage of PARP and caspase-3) suggest that FLLL12 is 5-10-fold more potent than curcumin against a panel of premalignant and malignant lung cancer cell lines, depending on the cell line. Moreover, FLLL12 induced the expression of death receptor-5 (DR5). Ablation of the expression of the components of the extrinsic apoptotic pathway (DR5, caspase-8 and Bid) by siRNA significantly protected cells from FLLL12-induced apoptosis (p < 0.05). Analysis of mRNA expression revealed that FLLL-12 had no significant effect on the expression of DR5 mRNA expression. Interestingly, inhibition of global phosphatase activity as well as protein tyrosine phosphatases (PTPs), but not of alkaline phosphatases, strongly inhibited DR5 expression and significantly inhibited apoptosis (p < 0.05), suggesting the involvement of PTPs in the regulation of DR5 expression and apoptosis. We further showed that the apoptosis is independent of p53 and p73. Taken together, our results strongly suggest that FLLL12 induces apoptosis of lung cancer cell lines by posttranscriptional regulation of DR5 through activation of protein tyrosine phosphatase(s).

Curcumin induces apoptosis of upper aerodigestive tract cancer cells by targeting multiple pathways.

Curcumin, a natural compound isolated from the Indian spice "Haldi" or "curry powder", has been used for centuries as a traditional remedy for many ailments. Recently, the potential use of curcumin in cancer prevention and therapy urges studies to uncover the molecular mechanisms associated with its anti-tumor effects. In the current manuscript, we investigated the mechanism of curcumin-induced apoptosis in upper aerodigestive tract cancer cell lines and showed that curcumin-induced apoptosis is mediated by the modulation of multiple pathways such as induction of p73, and inhibition of p-AKT and Bcl-2. Treatment of cells with curcumin induced both p53 and the related protein p73 in head and neck and lung cancer cell lines. Inactivation of p73 by dominant negative p73 significantly protected cells from curcumin-induced apoptosis, whereas ablation of p53 by shRNA had no effect. Curcumin treatment also strongly inhibited p-AKT and Bcl-2 and overexpression of constitutively active AKT or Bcl-2 significantly inhibited curcumin-induced apoptosis. Taken together, our findings suggest that curcumin-induced apoptosis is mediated via activating tumor suppressor p73 and inhibiting p-AKT and Bcl-2.