Burden of disease predicts response to isolated limb infusion with melphalan and actinomycin D in melanoma.

BACKGROUND: Isolated limb infusion (ILI) with melphalan is a minimally invasive, effective treatment for in transit melanoma. We hypothesized that burden of disease (BOD) would correlate to treatment response. METHODS: We retrospectively analyzed a prospectively collected database from two academic centers. BOD was stratified as high or low (low ≤ 10 lesions, none >2 cm). Response rates were measured 3 months post-ILI. Multivariable analysis (MV) was used to evaluate the association between the response and BOD. Kaplan-Meier methods with log-rank tests and MV Cox proportional hazard models were used to analyze overall survival (OS) and progression free survival (PFS). RESULTS: Sixty (38 %) patients had low and 100 (62 %) high BOD. Patients with low BOD had an overall response rate (ORR) of 73 % with 50 % CR, compared with an ORR of 47 % with 24 % CR in patients with high BOD (p = 0.002). MV analysis of preoperative, intraoperative, and postoperative parameters showed no significant impact on 3-month response. Patients with a CR at 3 months demonstrated improved PFS over the remainder of the cohort, but OS was similar. Low BOD patients had an increased median PFS of 6.9 versus 3.8 months (p = 0.047) and a increased median OS of 38.4 versus 30.9 months (p = 0.146). CONCLUSIONS: Lower BOD is associated with an increased ORR and CR rate with statistically significantly improved PFS in patients undergoing ILI for in transit extremity melanoma. BOD provides useful prognostic information for patient counseling and serves as a marker to stratify patient risk groups.

Pegylated arginine deiminase synergistically increases the cytotoxicity of gemcitabine in human pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma has proven to be one of the most chemo-resistant among all solid organ malignancies. Several mechanisms of resistance have been described, though few reports of strategies to overcome this chemo-resistance have been successful in restoring sensitivity to the primary chemotherapy (gemcitabine) and enter the clinical treatment arena. METHODS: We examined the ability of cellular arginine depletion through treatment with PEG-ADI to alter in vitro and in vivo cytotoxicity of gemcitabine. The effect on levels of key regulators of gemcitabine efficacy (e.g. RRM2, hENT1, and dCK) were examined. RESULTS: Combination of PEG-ADI and gemcitabine substantially increases growth arrest, leading to increased tumor response in vivo. PEG-ADI is a strong inhibitor of the gemcitabine-induced overexpression of ribonucleotide reductase subunit M2 (RRM2) levels both in vivo and in vitro, which is associated with gemcitabine resistance. This mechanism is through the abrogation of the gemcitabine-mediated inhibitory effect on E2F-1 function, a transcriptional repressor of RRM2. CONCLUSION: The ability to alter gemcitabine resistance in a targeted manner by inducing metabolic stress holds great promise in the treatment of advanced pancreatic cancer.

Role of autophagy in apoptotic regulation by Akt in pancreatic cancer.

BACKGROUND/AIM: The Akt signaling pathway mediates a potent anti-apoptotic signal in pancreatic cancer and inhibition of this pathway has become an attractive mechanism to increase the efficacy of traditional chemotherapies. Autophagy is a lysosomal catabolic pathway by which eukaryotic cells recycle macromolecules and organelles. Although autophagy may function as a survival mechanism under metabolic stress conditions, it also serves as an alternate route to programmed cell death distinct from apoptosis. In the present study, we examined the role of autophagy in Akt-mediated regulation of cell death in pancreatic cancer. MATERIALS AND METHODS: Mia-PaCa-2 and PANC-1 human pancreatic cancer cell lines were used in our experiments. The small-molecule inhibitor A-443654 was used to inhibit Akt, and rapamycin was used to inhibit mTOR. Autophagy was inhibited with Chloroquine and 3-methyladenine. Autophagy was assessed by immunoblotting for light chain-3 (LC-3) processing as well as fluorescence microscopy for autophagosome formation following transfection with a LC-3/GFP construct. Cell death was determined by fluorescence-activated cell sorting (FACS) with quantitation of the sub-G0 content. RESULTS: Inhibition of either Akt or mTOR induced autophagy; inhibition of Akt but not of mTOR led to traditional caspase-mediated apoptosis. When autophagy was inhibited, cell death was abrogated following Akt, but not mTOR, inhibition. CONCLUSION: The Akt signaling pathway regulates both autophagy and apoptosis through divergent pathways; mTOR mediates autophagy signaling but appears to be un-involved in cell death. Autophagy appears to play a role in the regulation of cell survival by Akt, but only when proximal signaling pathways not involving mTOR are simultaneously activated.

The role of Akt activation in the response to chemotherapy in pancreatic cancer.

UNLABELLED: The PI3K/Akt signaling pathway is constitutively activated in some pancreatic cancers; when activated, it inhibits chemotherapy-mediated apoptosis. We examined whether Akt activity correlates with apoptotic resistance to chemotherapy in pancreatic cancer. MATERIALS AND METHODS: A panel of human pancreatic cancer cells was evaluated for basal Akt activity as well as response to three chemotherapies. Chemotherapy-induced cell death was evaluated following either up- or down-regulation of Akt activity. Evaluation of phosphorylation of p21Cip/Waf1, a downstream target of Akt, was also evaluated. RESULTS: There was a broad distribution among pancreatic cancer cell lines by Akt activity, as well as sensitivity to the three chemotherapeutic agents with no apparent correlation. Phosphorylation of p21Cip/Waf1, but not change in total levels, correlated with the chemosensitizing effect of Akt inhibition to paclitaxel. CONCLUSIONS: Basal Akt activity does not appear to be a useful predictor for selection of pancreatic cancers in targeting Akt to broadly induce chemosensitivity.

Targeting Bcl-2-mediated cell death as a novel therapy in pancreatic cancer.

BACKGROUND: Bcl-2 is an essential regulator of programmed cell death (PCD). Overexpression of Bcl-2 is common in pancreatic cancer; the high levels have been shown to correlate with resistance to PCD. This resistance is mediated by binding of Bcl-2 via its BH-3 domain to diverse proteins, including the Bax/Bak family members, various protein kinases, and beclin 1, which are involved in regulation of autophagy (type II PCD). Small molecule inhibitors of BH-3-mediated binding of Bcl-2 have been recently developed, although no investigation has been conducted in pancreatic cancer, a malignancy characterized by extreme resistance to PCD. METHODS: The effect of the Bcl-2 binding inhibitor A-779024 on PCD was assessed by fluorescence activated cell sorting; the effect on Bcl-2 and other PCD-related proteins was analyzed by immunoblotting. Induction of autophagy was determined by fluorescence microscopy using a stably transfected GFP-LC3 construct to visualize autophagosome formation. Co-localization of Bcl-2 with binding partners regulating PCD was examined by immunoprecipitation and confocal immunofluorescent microscopy. RESULTS: A-779024 induced PCD in a dose- and time-dependent fashion. No change was seen in the protein levels of Bcl-2, Bax, Bcl-XL, or Mcl-1. Contrary to prediction, A-779024 was ineffective at inducing autophagy in these cells. Co-localization studies demonstrated that Bcl-2 was not bound to beclin 1 and, therefore, treatment with A-779024 could not induce release of beclin 1 and initiation of autophagy. CONCLUSIONS: Disruption of Bcl-2 activity using the small molecule inhibitor A-779024 induces apoptotic but not autophagic PCD. This approach may be a novel therapy, either alone or in combination with other treatments such as chemotherapy or autophagy modulating agents in pancreatic cancer.

Surgery in the patient with liver disease.

Liver dysfunction is a prominent entity in Western medicine that has historically affected patients suffering from chronic viral or alcoholic hepatitis. The incidence of these conditions has not changed dramatically in recent years but the overall number of patients with liver dysfunction has increased considerably with the emergence of the obesity epidemic. Nonalcoholic fatty liver disease (NAFLD) has become increasingly recognized as the most common cause of chronic liver disease in the United States. Although the rate of progression of NAFLD to overt cirrhosis is low, the high prevalence of this condition, combined with the moderate degree of liver dysfunction it engenders, has resulted in a significant increase in the number of patients with liver disease that can be encountered by a surgical practice. Any degree of clinically evident liver disease in a prospective surgical patient should raise concern for the entire surgical team. This particularly applies to intraabdominal surgery whereby the presence of hepatomegaly, portal hypertension, variceal bleeding, and ascites can turn even the most routine operation into a morbid and life-threatening procedure. Nonabdominal surgery avoids some of the technical challenges presented by liver disease but the anesthetic management of a cirrhotic patient still makes any operation potentially more dangerous. In this article, approaches to minimize the risk when surgery becomes necessary in the presence of liver disease are discussed.

Surgery in the patient with liver disease.

Liver dysfunction is a prominent entity in Western medicine that has historically affected patients suffering from chronic viral or alcoholic hepatitis. The incidence of these conditions has not changed dramatically in recent years but the overall number of patients with liver dysfunction has increased considerably with the emergence of the obesity epidemic. Nonalcoholic fatty liver disease (NAFLD) has become increasingly recognized as the most common cause of chronic liver disease in the United States. Although the rate of progression of NAFLD to overt cirrhosis is low, the high prevalence of this condition, combined with the moderate degree of liver dysfunction it engenders, has resulted in a significant increase in the number of patients with liver disease that can be encountered by a surgical practice. Any degree of clinically evident liver disease in a prospective surgical patient should raise concern for the entire surgical team. This particularly applies to intraabdominal surgery whereby the presence of hepatomegaly, portal hypertension, variceal bleeding, and ascites can turn even the most routine operation into a morbid and life-threatening procedure. Nonabdominal surgery avoids some of the technical challenges presented by liver disease but the anesthetic management of a cirrhotic patient still makes any operation potentially more dangerous. In this article, approaches to minimize the risk when surgery becomes necessary in the presence of liver disease are discussed.

Lys40 but not Arg143 influences selectivity of angiotensin conversion by human alpha-chymase.

Human alpha-chymase is an efficient angiotensin (AT) converting enzyme, selectively hydrolyzing AT I at Phe8 to generate bioactive AT II, which can promote cardiac hypertrophy, vascular stenosis, and hypertension. Some related enzymes, such as rat beta-chymase 1, are much less selective, destroying AT by cleaving at Tyr4. Comparisons of chymase structure and activity led to speculation that interaction between AT and the side chain of Lys40 or Arg143 accounts for the human enzyme's marked preference for Phe8 over Tyr4. To test these hypotheses, we compared AT hydrolysis by wild-type chymase with that by mutants changing Lys40 or Arg143 to neutral residues. Lys40 was exchanged for alanine, the residue found in canine alpha- and rat beta-chymase 1, the latter being dramatically less selective for hydrolysis at Phe8. Arg143 was exchanged for glutamine found in rat beta-chymase 1. The Lys40Ala mutant is a dog-like enzyme retaining strong preference for Phe8 but with Tyr4 hydrolytic rates enhanced 16-fold compared to wild-type human enzyme. Thus, of 40 residues mismatched between dog and human enzymes, a single residue accounts for most of the difference in specificity between them. The Arg143Gln mutant, contrary to prediction, remains highly Phe8-selective. Therefore, Lys40, but not Arg143, contributes to human chymase's remarkable preference for AT II generation over destruction.