Activity Of Gemcitabine Research Articles

Safety, tolerability and antitumour activity of LY2780301 (p70S6K/AKT inhibitor) in combination with gemcitabine in molecularly selected patients with advanced or metastatic cancer: a phase IB dose escalation study

BackgroundLY2780301, a dual inhibitor of protein kinase B (AKT) and the downstream effector p70 ribosomal protein S6 kinase (p70S6K), may inhibit progression in tumours relying on phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signalling pathway activation. This phase IB trial investigated the maximum tolerated dose (MTD), dose-limiting toxicities (DLTs), safety, pharmacokinetics (PK) and antitumour activity of LY2780301 plus gemcitabine in patients with advanced/metastatic solid tumours. MethodsThis was a non-randomised, open-label, dose escalation and dose expansion trial. Patients harbouring molecular alterations of the PI3K/AKT/mTOR pathway received once daily (QD) oral LY2780301 (400 or 500 mg) in combination with intravenous gemcitabine (750 or 1000 mg/m2) on days 1, 8 and 15 of a 28-d cycle. Dose escalation followed a 3 + 3 design. Assessments included adverse events (AEs), PK and preliminary antitumour activity. ResultsFifty patients (median age, 53 years; 74% female) predominantly with mutations/amplifications of PI3K (60%) and phosphatase and tensin homologue (PTEN) gene/protein inactivation (42%) were treated for up to 14 cycles. The MTD was LY2780301 500 mg QD with gemcitabine 750 mg/m2. DLTs during cycle 1 were grade IV thrombocytopenia, grade III skin rash and grade III increase in alkaline phosphatase, gamma glutamyltransferase and alanine aminotransferase, occurring in one patient each. Most common AEs were anaemia (84%), fatigue (84%), transaminase increase (74%), thrombocytopenia (74%), nausea/vomiting (70%), neutropenia (68%) and lymphopenia (56%). Among the efficacy-evaluable population, two patients (5%) had a partial response; the disease control rate was 74% at cycle 2. ConclusionsAddition of LY2780301 to gemcitabine showed manageable toxicity and encouraging antitumour activity in patients with molecular alterations of the PI3K/AKT/mTOR pathway. Clinical trial registration numberNCT02018874.

Antiviral activity of gemcitabine against human rhinovirus in vitro and in vivo

Rhinovirus, a major causative agent of the common cold, is associated with exacerbation of asthma and chronic obstructive pulmonary disease. Currently, there is no antiviral treatment or vaccine for human rhinovirus (HRV). Gemcitabine (2′,2′-difluorodeoxycytidine, dFdC) is a deoxycytidine analog with antiviral activity against rhinovirus, as well as enterovirus 71, in vitro. However, the antiviral effects of gemcitabine in vivo have not been investigated. In the current study, we assessed whether gemcitabine mediated antiviral effects in the murine HRV infection model. Intranasal administration of gemcitabine significantly lowered pulmonary viral load and inflammation by decreasing proinflammatory cytokines, including TNF-α and IL-1β, and reduction in the number of lung-infiltrating lymphocytes. Interestingly, we found that the addition of UTP and CTP significantly attenuated the antiviral activity of gemcitabine. Thus the limitation of UTP and CTP by the addition of gemcitabine may inhibit the viral RNA synthesis. These results suggest that gemcitabine, an antineoplastic drug, can be repositioned as an antiviral drug to inhibit HRV infection.

Activation of a c-Jun N-terminal kinase-mediated autophagy pathway attenuates the anticancer activity of gemcitabine in human bladder cancer cells.

The role of autophagy in the anticancer activity of gemcitabine (GEM) in bladder cancer is unclear. The aim of this study is to determine whether GEM activates autophagy, the role of autophagy in the anticancer activity of GEM, and the underlying mechanism by which GEM induces autophagy. Human bladder cancer cell lines T24 and BIU87 were treated with GEM in vitro. Cell viability was measured using the Cell Counting Kit-8 assay. Apoptosis was detected by annexin V assay and western blot. Autophagy was measured by western blot and transmission electron microscopy. c-Jun N-terminal kinase (JNK) activation was detected by western blot. Chemical inhibitors were used for intervention of JNK and autophagy. GEM killed bladder cancer cells, which was associated with apoptosis induction. Autophagy was effectively activated by GEM. Suppressing autophagy in GEM-treated cells significantly decreased cell viability, which was associated with increased apoptosis. GEM-induced JNK activation and suppressed B-cell lymphoma 2 expression. The JNK inhibitor SP600125 inhibited GEM-induced autophagy activation and increased GEM's cytotoxicity. GEM kills bladder cancer cells through apoptosis. Meanwhile, JNK-mediated autophagy was activated, which protects the cells against apoptosis. Therefore, inhibition of autophagy could be exploited to enhance the anticancer efficacy of GEM for treating bladder cancer.

Abstract 4731: Phospho-akt: a potential resistance marker to chemotherapy and a therapy-target to restore sensitivity in pancreatic cancer

Abstract Oncogenic KRAS signaling is the main driving force behind pancreatic ductal adenocarcinoma (PDAC); however, targeting this pathway has proven to be difficult. Conversely, the PI3K/Akt pathway represents an exciting new target, because it has been associated with poor prognosis and chemoresistance, and several inhibitors are under development. In particular, perifosine prevents Akt translocation to the cell membrane, while MK-2206 is an Akt allosteric inhibitor and BEZ-235 is a dual PI3K/mTOR inhibitor. Therefore, we investigated the prognostic role of phospho (p)-Akt in PDAC tissues, as well as the molecular mechanisms underlying the interaction of Akt inhibitors with gemcitabine, using PDAC cells, primary cultures and spheroids. Immunohistochemistry of tissue microarrays with specimens from radically-resected patients (n=100) revealed a correlation between high p-Akt1 expression and worse outcome. Patients with low p-Akt expression (as detected by digital scoring) had a median overall survival (OS) of 16.2 months (95% CI, 14.8-20.1), while patients with high expression had a median OS of 12.0 months (95% CI, 9.0-14.9, P=0.03). Parallel immunocytochemistry studies revealed high expression levels in LPC028 primary cells, while LPC006 were characterized by low p-Akt1. Akt inhibitors reduced cancer cell growth in monolayers and spheroids, and synergistically enhanced the antiproliferative activity of gemcitabine in LPC028 (e.g., combination index CI of 0.2, in the gemcitabine-perifosine combination for 72h, at fixed IC50 ratio), while this combination was antagonistic in LPC006 cells. The synergistic effect was paralleled by a 5-fold reduction in the expression of the main gemcitabine target ribonucleotide reductase. Inhibition of Akt decreased cell migration and invasion, which was additionally reduced by the combination with gemcitabine. However, the combination of Akt inhibitors with gemcitabine increased apoptosis, associated with induction of caspase-3/6/8/9, PARP and BAD, and inhibition of Bcl-2 and NF-kB in LPC028, but not in LPC006 cells. The Akt signaling is involved in the expression/localization of the key glucose transporter Glut1, and increased glucose metabolism was associated to resistance to axitinib (Hudson et al, Cell Death Dis 2014). Remarkably, the resistant LPC006 cells were characterized by overexpression of Glut1, which was not reduced after exposure to Akt inhibitors. However, the novel Glut1 inhibitor PGL13 enhanced perifosine and perifosine/gemcitabine-induced cell death. In conclusion, our findings support the analysis of phospho-Akt1 expression as both a prognostic and a predictive biomarker, for the rational development of new combination therapies targeting the Akt pathway in PDAC. Finally, inhibition of Glut1 might overcome resistance to these therapies and warrants further studies. Citation Format: Daniela Massihnia, Amir Avan, Niccola Funel, Carlotta Granchi, Filippo Minutolo, Leticia Leon, Godefridus Peters, Elisa Giovannetti. Phospho-akt: a potential resistance marker to chemotherapy and a therapy-target to restore sensitivity in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4731. doi:10.1158/1538-7445.AM2017-4731

Gemcitabine-vitamin E conjugates: Synthesis, characterization, entrapment into nanoemulsions, and in-vitro deamination and antitumor activity

Gemcitabine is the first line therapy for pancreatic cancer. It is, however, extensively metabolized to the inactive form by deamination enzymatic reaction. Conjugation of gemcitabine with fatty acids on its 4-amino group was found to protect it from deamination deactivation reaction. The objective of the present study was to test the in-vitro anticancer activity of gemcitabine conjugated to the γ-tocotrienol isomer of vitamin E against pancreatic tumor cells. This objective was based on reported studies in which it was demonstrated that free tocotrienol isomers of vitamin E can potentiate the anticancer activity of gemcitabine. To accomplish this objective, a full synthesis scheme for gemcitabine conjugation to fatty acids (stearic and linoleic) and the tocopherol and tocotrienol isomers of vitamin E (α-T and γ-T3) was presented. The conjugates were characterized by 1H NMR and mass spectrometry analysis and tested for their susceptibility to deamination. Also discussed is the impact of entrapping the conjugates into nanoemulsions on the physiochemical properties of the delivery system and the in vitro anticancer activity of gemcitabine against Bx-PC-3 and PNAC-1 pancreatic cancer cells. In-vitro enzymatic deamination study showed that the γ-T3 conjugate of gemcitabine was least affected by deamination deactivation reaction when compared with the free and conjugated gemcitabine in solution. Furthermore, in-vitro cytotoxicity study demonstrated that entrapment of gemcitabine-lipid conjugates into nanoemulsions significantly enhanced their anticancer activity when compared to the free drug. It was concluded that conjugation to the γ-T3 isomer is a viable option for gemcitabine delivery and is worthy of further investigation.

Enhanced antitumor activity of gemcitabine by polysaccharide-induced NK cell activation and immune cytotoxicity reduction in vitro/vivo

The polysaccharide SEP has been reported to activate NK and T cells via TLR2/4. Here, the combination of gemcitabine (GEM) and SEP against HepG-2 was investigated. SEP apparently enhanced antitumor activity of gemcitabine against liver cancer through stimulating NKG2D and DAP10/Akt pathway to activate NK cells. The NKG2D upregulation could improve the sensitivity of NK-92 cells targeting to its ligand MICA expressed on HepG-2 cells. Meanwhile, GEM up-regulated MICA expression and attenuated soluble MICA secretion through inhibiting ADAM10 expression, which in turn enhanced the cytotoxicity of NK-92 cells against cancer cells. SEP remarkably enhanced GEM antitumor activity with an inhibitory rate of 79.1% in an H22-bearing mouse model. Moreover, SEP reversed atrophy and apoptosis caused by GEM in both spleen and bone marrow through suppressing ROS secretion in vivo. The data indicated that the combination of SEP and GEM is a potential chemo-immunotherapy strategy for liver cancer treatment clinically.

Cellular uptake and anti-tumor activity of gemcitabine conjugated with new amphiphilic cell penetrating peptides.

Gemcitabine (Gem) is used as a single agent or in combination with other anticancer agents to treat many types of solid tumors. However, it has many limitations such as a short plasma half-life, dose-limiting toxicities and drug resistance. Cell-penetrating peptides (CPPs) are short peptides which may deliver a large variety of cargo molecules into the cancerous cells. The current study was designed to evaluate the antiproliferative activity of gemcitabine chemically conjugated to CPPs. The peptides were synthesized using solid phase synthesis procedure. The uptake efficiency of CPPs into cells was examined by flow cytometry and fluorescent microscopy. The synthesized peptides were chemically conjugated to Gem and the in vitro cytotoxicity of conjugates was tested by MTT assay on A594 cell line. According to the obtained results, cellular uptake was increased with increasing the concentration of CPPs. On the other hand the coupling of Gem with peptides containing block sequence of arginine (R5W3R4) and some alternating sequences (i.e. [RW]6 and [RW]3) exhibited improved antitumor activity of the drug. The findings in this study support the advantages of using cell-penetrating peptides for improving intracellular delivery of Gem into tumor as well as its activity.

Gemcitabine anti-proliferative activity significantly enhanced upon conjugation with cell-penetrating peptides

Gemcitabine proven efficiency against a wide range of solid tumors and undergoes deamination to its inactive uridine metabolite, which underlies its low bioavailability, and tumour resistance was also associated with nucleoside transporter alterations. Hence, we have conjugated gemcitabine to cell-penetrating peptides (CPP), in an effort to both mask its aniline moiety and facilitate its delivery into cancer cells. Two CPP-drug conjugates have been synthesized and studied regarding both the time-dependent kinetics of gemcitabine release and their anti-proliferative activity on three different human cancer cell lines. Results obtained reveal a dramatic increase in the anti-proliferative activity of gemcitabine in vitro, upon conjugation with the CPPs. As such, CPP-gemcitabine conjugates emerge as promising leads for cancer therapy.

HSPA5 Regulates Ferroptotic Cell Death in Cancer Cells.

Ferroptosis is a form of regulated cell death driven by oxidative injury promoting lipid peroxidation, although detailed molecular regulators are largely unknown. Here, we show that heatshock 70-kDa protein 5 (HSPA5) negatively regulates ferroptosis in human pancreatic ductal adenocarcinoma (PDAC) cells. Mechanistically, activating transcription factor 4 (ATF4) resulted in the induction of HSPA5, which in turn bound glutathione peroxidase 4 (GPX4) and protected against GPX4 protein degradation and subsequent lipid peroxidation. Importantly, the HSPA5-GPX4 pathway mediated ferroptosis resistance, limiting the anticancer activity of gemcitabine. Genetic or pharmacologic inhibition of the HSPA5-GPX4 pathway enhanced gemcitabine sensitivity by disinhibiting ferroptosis in vitro and in both subcutaneous and orthotopic animal models of PDAC. Collectively, these findings identify a novel role of HSPA5 in ferroptosis and suggest a potential therapeutic strategy for overcoming gemcitabine resistance. Cancer Res; 77(8); 2064-77. ©2017 AACR.

Long-term results and recurrence patterns from SCALOP: a phase II randomised trial of gemcitabine- or capecitabine-based chemoradiation for locally advanced pancreatic cancer

Background:SCALOP, a randomised, phase II trial, tested the activity and safety of gemcitabine (GEM)-based and capecitabine (CAP)-based chemoradiation (CRT) for locally advanced pancreatic cancer (LAPC). Here we present the long-term outcomes.Methods:Eligibility: histologically proven LAPC ⩽7 cm. Following 12 weeks of induction GEMCAP chemotherapy (three cycles: GEM 1000 mg m−2 days 1, 8, 15; CAP 830 mg m−2 days 1–21 q28 days) patients with stable/responding disease, tumour ⩽6 cm, and WHO Performance Status 0–1 were randomised to receive one cycle GEMCAP followed by CAP (830 mg m−2 b.d. on weekdays only) or GEM (300 mg m−2 weekly) with radiation (50.4 Gy per 28 fractions).Results:One-hundred fourteen patients (28 UK centres) were registered between 24 December 2009 and 25 October 2011, and 74 were randomised (CAP-RT=36; GEM-RT=38). At the time of this analysis, 105 of the 114 patients had died and the surviving 9 patients had been followed up for a median of 10.9 months (IQR: 2.9–18.7). Updated median OS was 17.6 months (95% CI: 14.6–22.7) in the CAP-CRT arm and 14.6 months (95% CI: 11.1–16.0) in the GEM-CRT arm (intention-to-treat adjusted hazard ratio (HR): 0.68 (95% CI: 0.38–1.21, P=0.185)); median progression-free survival (PFS) was 12.0 months (95% CI: 10.0–15.2) in the CAP-CRT arm and 10.4 months (95% CI: 8.8–12.7) in the GEM-CRT arm (intention-to-treat adjusted HR: 0.60 (95% CI: 0.32–1.14, P=0.120)). In baseline multivariable model, age ⩾65 years, better performance status, CA19.9<613 IU l−1, and shorter tumour diameter predicted improved OS. CAP-CRT, age ⩾65 years, better performance status, CA19.9 <46 IU ml−1 predicted improved OS and PFS in the pre-radiotherapy model. Nine-month PFS was highly predictive of OS.Conclusions:CAP-CRT remains the superior regimen. SCALOP showed that patients with CA19.9 <46 IU ml−1 after induction chemotherapy are more likely to benefit from CRT.

Curcumin and its cyclohexanone analogue inhibited human Equilibrative nucleoside transporter 1 (ENT1) in pancreatic cancer cells

Our group investigated combining the phytochemical curcumin and gemcitabine in a liposome, to improve gemcitabine's activity against pancreatic tumours. While optimising the curcumin: gemcitabine ratio for co-encapsulation, we found that increasing curcumin concentrations relative to gemcitabine resulted in antagonistic interactions. As curcumin is a promiscuous transporter inhibitor; we suspected that increased resistance occurred via inhibition of Equilibrative nucleoside transporter 1 (ENT1)-mediated gemcitabine uptake. To test our hypothesis, we determined whether curcumin and a related analogue, 2,6-bis((3-methoxy-4-hydroxyphenyl)methylene)-cyclohexanone (or A13), inhibited ENT1-mediated accumulation of [3H]uridine and [3H]gemcitabine into pancreatic cancer cells. We then confirmed the inhibition of gemcitabine accumulation by investigating whether curcumin/A13 could increase gemcitabine resistance in growth inhibition assays. We found that curcumin and A13 concentration-dependently inhibited the ENT1-mediated accumulation of both uridine and gemcitabine in MIA PaCa-2 and PANC-1 cells. We also found that non-toxic concentrations of curcumin and A13 significantly increased the resistance of both cell lines to gemcitabine. Increased resistance only occurred when curcumin/A13 was co-incubated with gemcitabine, and not with sequential exposure (i.e., curcumin first, followed by gemcitabine, or vice versa). We also found that the curcumin analogue (3E,5E)−3,5-bis[(2-fluorophenyl)methylene]−4-piperidinone (or EF24) did not inhibit gemcitabine accumulation, making it more suitable in combinations than curcumin/A13. From these results, we concluded that curcumin and A13 are inhibitors of the ENT1 transporter, but only at high concentrations (2–20µM). Curcumin is unlikely to inhibit gemcitabine uptake in tumours but may interfere with the oral absorption of ENT1 substrates due to high gut concentrations readily achievable from over-the-counter tablets/capsules.

Development of bioluminescent chick chorioallantoic membrane (CAM) models for primary pancreatic cancer cells: a platform for drug testing

The aim of the present study was to develop chick-embryo chorioallantoic membrane (CAM) bioluminescent tumor models employing low passage cell cultures obtained from primary pancreatic ductal adenocarcinoma (PDAC) cells. Primary PDAC cells transduced with lentivirus expressing Firefly-luciferase (Fluc) were established and inoculated onto the CAM membrane, with >80% engraftment. Fluc signal reliably correlated with tumor growth. Tumor features were evaluated by immunohistochemistry and genetic analyses, including analysis of mutations and mRNA expression of PDAC pivotal genes, as well as microRNA (miRNA) profiling. These studies showed that CAM tumors had histopathological and genetic characteristic comparable to the original tumors. We subsequently tested the modulation of key miRNAs and the activity of gemcitabine and crizotinib on CAM tumors, showing that combination treatment resulted in 63% inhibition of tumor growth as compared to control (p < 0.01). These results were associated with reduced expression of miR-21 and increased expression of miR-155. Our study provides the first evidence that transduced primary PDAC cells can form tumors on the CAM, retaining several histopathological and (epi)genetic characteristics of original tumors. Moreover, our results support the use of these models for drug testing, providing insights on molecular mechanisms underlying antitumor activity of new drugs/combinations.

Therapeutic Potential of Curcumin in Treatment of Pancreatic Cancer: Current Status and Future Perspectives.

Pancreatic cancer is among the leading cause of deaths due to cancer with extremely poor prognosis. Gemcitabine is being used in the treatment of patient with pancreatic ductal adenocarcinoma (PDAC), although, the response rate is bellow 12%. A recent phase III trial revealed that FOLFIRINOX could be an option for the treatment of metastatic PDAC patients, although it is associated with increased toxicity. Therefore, identification of novel agents that either improves gemcitabine activity, within novel combinatorial approaches, or with a better efficacy than gemcitabine is warranted. The antitumor activity of curcumin in several tumors, including prostate, breast and colorectal cancers have investigated. A recent phase II trial explored the effects of curcumin in advanced pancreatic cancer patient. They found that oral curcumin was well tolerated. Another trial showed the activity of 8,000 mg of curcumin in combination with gemcitabine in patients with advanced pancreatic cancer. This review summarizes the current knowledge about possible molecular mechanisms of curcumin in PDAC with particular emphasis on preclinical/clinical studies in pancreatic cancer treatment. J. Cell. Biochem. 118: 1634-1638, 2017. © 2017 Wiley Periodicals, Inc.

All-trans retinoic acid enhances gemcitabine cytotoxicity in human pancreatic cancer cell line AsPC-1 by up-regulating protein expression of deoxycytidine kinase

We previously showed that gemcitabine resistance in pancreatic cancer chemotherapy correlates with suppressed expression of deoxycytidine kinase (dCK), which catalyzes the rate-limiting step of gemcitabine activation. The purpose of the present study was to find a drug that might be useful to enhance the cytotoxicity of gemcitabine by increasing dCK expression in gemcitabine-resistant human pancreatic cancer cell line AsPC-1. Screening of 40 prescription drugs identified 35 with no intrinsic cytotoxicity towards AsPC-1 cells. When AsPC-1 cells were pre-incubated with these drugs and then incubated with gemcitabine, we found that all-trans retinoic acid (ATRA) significantly decreased the viability by 28% compared with that of non-treated cells. Luciferase assay showed that ATRA transactivated the DCK promoter in AsPC-1 cells by about 2-fold compared with the untreated control, and an increase of dCK protein expression was confirmed by immunoblotting. ATRA decreased the half-maximal inhibitory concentration (IC50) of gemcitabine by 2.8-fold (ATRA-non-treated cells, 28.8nM; ATRA-treated cells, 10.0nM). The ATRA concentration of 0.03μM was sufficient to enhance gemcitabine cytotoxicity, and the effect was well maintained in the concentration range from 0.03 to 50μM. These results indicate that ATRA enhances gemcitabine cytotoxicity by increasing dCK expression in gemcitabine-resistant human pancreatic cancer cells.

Preclinical antitumor activity of nanoliposomal irinotecan (Nal-IRI, MM-398) and utilization as a foundation of front-line pancreatic cancer regimens.

336 Background: Nanoliposomal irinotecan (nal-IRI, MM-398) recently gained approval in combination with 5-fluorouracil/leucovorin (5-FU/LV) in post-gemcitabine metastatic pancreatic ductal adenocarcinoma (PDAC) based on the extended survival and manageable safety profile observed in the Phase 3 NAPOLI-1 trial. Preclinically, we have previously demonstrated the anti-tumor activity of nal-IRI with 5-FU and oxaliplatin, standard of care agents in first-line PDAC, and are currently investigating this combination in patients with previously untreated metastatic PDAC in a Phase 2 clinical trial (NCT02551991). Herein, we further evaluate nal-IRI as a potential backbone of first-line metastatic PDAC by assessing the preclinical anti-tumor activity of nal-IRI relative to, and in combination with, gemcitabine and nanoparticle albumin-bound-paclitaxel (nab-P). Methods: Nal-IRI tumor metabolite (CPT-11 and SN-38) levels were measured in mice treated with nal-IRI in combination with gemcitabine or nab-P. Anti-tumor activity and tolerability of nal-IRI, 5-FU, gemcitabine and nab-P monotherapies and combinations were evaluated using pancreatic cancer cell line (ASPC-1 and CFPAC-1)-derived xenograft models, as well as a panel of five patient-derived xenograft models. Results: Administration of gemcitabine or nab-P prior to or simultaneously with nal-IRI resulted in unchanged or increased nal-IRI deposition, as measured by tumor CPT-11 and SN-38 levels at 24 hours post-injection. Moreover, in both cell line-derived and patient-derived xenograft models of PDAC, nal-IRI monotherapy demonstrated comparable or improved anti-tumor activity relative to gemcitabine or nab-P monotherapies. Further, nal-IRI consistently improved tumor growth inhibition and survival when used in combination with either 5-FU, gemcitabine and/or nab-P, relative to the combination of gemcitabine plus nab-P. All treatments were well-tolerated in these preclinical models. Conclusions: These findings illustrate the compatibility and therapeutic potential of nal-IRI as a foundation of first-line PDAC combination regimens, and warrant clinical evaluation.

Total Syntheses and Biological Activities of Vinylamycin Analogues.

Natural depsipeptide vinylamycin was reported to be an antibiotic previously. Herein we report vinylamycin to be active against K562 leukemia cells (IC50 = 4.86 μM) and be unstable in plasma (t1/2 = 0.54 h). A total of 24 vinylamycin analogues with modification of the OH group and chiral centers were generated via a combinatorial approach. The lead compound 1a was subsequently characterized as having the following: no antimicrobial activity, significantly higher plasma stability (t1/2 = 14.3 h), improved activity against K562 leukemia cells (IC50 = 0.64 μM), and up to 75% cell inhibition without significant toxicities in K562 cells xenograft zebrafish model. Furthermore, compound 1a maintained its activity against the breast cancer cell line MCF-7 under hypoxic conditions. In comparison, the activity of gemcitabine in the same hypoxic in vitro model of MCF-7 cells was 15-fold lower. Therefore, the present results demonstrate that 1a has great potential as an anticancer agent.

Phospho-Akt overexpression is prognostic and can be used to tailor the synergistic interaction of Akt inhibitors with gemcitabine in pancreatic cancer

BackgroundThere is increasing evidence of a constitutive activation of Akt in pancreatic ductal adenocarcinoma (PDAC), associated with poor prognosis and chemoresistance. Therefore, we evaluated the expression of phospho-Akt in PDAC tissues and cells, and investigated molecular mechanisms influencing the therapeutic potential of Akt inhibition in combination with gemcitabine.MethodsPhospho-Akt expression was evaluated by immunohistochemistry in tissue microarrays (TMAs) with specimens tissue from radically-resected patients (n = 100). Data were analyzed by Fisher and log-rank test. In vitro studies were performed in 14 PDAC cells, including seven primary cultures, characterized for their Akt1 mRNA and phospho-Akt/Akt levels by quantitative-RT-PCR and immunocytochemistry. Growth inhibitory effects of Akt inhibitors and gemcitabine were evaluated by SRB assay, whereas modulation of Akt and phospho-Akt was investigated by Western blotting and ELISA. Cell cycle perturbation, apoptosis-induction, and anti-migratory behaviors were studied by flow cytometry, AnnexinV, membrane potential, and migration assay, while pharmacological interaction with gemcitabine was determined with combination index (CI) method.ResultsImmunohistochemistry of TMAs revealed a correlation between phospho-Akt expression and worse outcome, particularly in patients with the highest phospho-Akt levels, who had significantly shorter overall and progression-free-survival. Similar expression levels were detected in LPC028 primary cells, while LPC006 were characterized by low phospho-Akt. Remarkably, Akt inhibitors reduced cancer cell growth in monolayers and spheroids and synergistically enhanced the antiproliferative activity of gemcitabine in LPC028, while this combination was antagonistic in LPC006 cells. The synergistic effect was paralleled by a reduced expression of ribonucleotide reductase, potentially facilitating gemcitabine cytotoxicity. Inhibition of Akt decreased cell migration and invasion, which was additionally reduced by the combination with gemcitabine. This combination significantly increased apoptosis, associated with induction of caspase-3/6/8/9, PARP and BAD, and inhibition of Bcl-2 and NF-kB in LPC028, but not in LPC006 cells. However, targeting the key glucose transporter Glut1 resulted in similar apoptosis induction in LPC006 cells.ConclusionsThese data support the analysis of phospho-Akt expression as both a prognostic and a predictive biomarker, for the rational development of new combination therapies targeting the Akt pathway in PDAC. Finally, inhibition of Glut1 might overcome resistance to these therapies and warrants further studies.

Metformin potentiates the anticancer activities of gemcitabine and cisplatin against cholangiocarcinoma cells in vitro and in vivo.

Metformin, an oral biguanide drug used to treat type2 diabetes, has displayed anticancer activities in several types of cancer cells. The combination of gemcitabine and cisplatin is the standard chemotherapy regimen for cholangiocarcinoma, but its benefit is limited. The present study aimed to investigate whether metformin could enhance the activities of gemcitabine and cisplatin against cholangiocarcinoma, and the underlying mechanisms. Metformin inhibited the proliferation of human cholangiocarcinoma RBE and HCCC-9810 cells and induced cell cycle arrest at the G0/G1phase by increasing the activation of AMP-activated protein kinase (AMPK) pathways. Metformin upregulated the expression of p21Waf1 and p27kip1, and downregulated the expression of cyclinD1, a key protein required for cell cycle progression. The combination of gemcitabine and cisplatin inhibited the proliferation and induced the apoptosis of human cholangiocarcinoma cells by inducing the phosphorylation of AMPK, downregulating cyclinD1, and activating caspase-3. Administration of metformin enhanced the efficacy of gemcitabine and cisplatin to suppress the growth of cholangiocarcinoma tumors established in experimental models by inhibiting cell proliferation and inducing cell apoptosis through their effects on AMPK, cyclinD1 and caspase-3. Given that metformin has been used to treat type2 diabetes patients for over half a century due to its superior safety profile, the results presented here indicate that metformin may be a potent agent for enhancing the efficacy of gemcitabine and cisplatin in the treatment of cholangiocarcinoma.

Discovery of a novel chimeric ubenimex–gemcitabine with potent oral antitumor activity

Herein, a novel mutual prodrug BC-A1 was discovered by integrating ubenimex and gemcitabine into one molecule. Biological characterization revealed that compound BC-A1 could maintain both the anti-CD13 activity of ubenimex and the cytotoxic activity of gemcitabine in vitro. Further characterization also demonstrated that compound BC-A1 exhibited significant anti-invasion and anti-angiogenesis effects in vitro. The preliminary stability test of BC-A1 revealed that it could release gemcitabine in vitro. The in vivo anti-tumor results in liver cancer showed that at the same dosage, oral administration of BC-A1 was as potent as intraperitoneal administration of gemcitabine. This warranted the further research and development of the orally active prodrug BC-A1 because gemcitabine can not be orally administrated in clinic.

Abstract B15: Patient-derived Pancreatic Adenocarcinoma cells: A new model system to define chemotherapy resistance mechanisms and to improve targeted personalized treatment

Abstract Background: Pancreatic adenocarcinoma (PA) is the fourth leading cause of cancer related death in the USA. Patients diagnosed with this disease can expect a 1-year survival rate of approximately 10%. One of the main reasons of the high mortality observed within PA is failure on first-line therapies. Current PA treatment involves Gemcitabine, a nucleoside analog that showed improvement in overall survival. Recently this drug has been used in combination with nab-paclitaxel, 5-FU, Erlotinib and MEK inhibitors. However, a growing number of patients have shown resistance to these regimes. A more comprehensive understanding of resistance mechanisms will enhance treatment choice and clinical responses, and this remains an area of intense investigation. However much of the data available on PA drug targets and efficacy come from commercially available pancreatic cell lines, a main limitation in PA research, as these lines do not accurately represent a given patients' tumor, underscoring the need for the development and use of patient-specific primary epithelial PA cells. In our current study we present the use of patient-derived primary PA cells as a model system for basic and translational research as well as personalized therapeutic approaches. Methods: Patients' biopsies were collected after surgery and long-term cultures of PA cells were established using the conditional reprogramming of cells (CRC) approach. To date, six primary lines have been continuous culture for over 6 month. KRAS and p53 sequencing verified the PA origin of both the patient sample and the matched CRC lines. CRC karyotyping was also performed to confirm the absence of normal cells as well as to validate the long-term genomic integrity of the lines in culture. In order to evaluate patient-specific differences in treatment response, the IC50's for gemcitabine, nab-paclitaxel (Abraxane) and the MEK inhibitor, Trametinib, were determined. To better understand treatment resistance mechanisms, new drug resistance approaches were developed and multiple drug-resistant clones per primary cell line were established, and their resistance verified. Gemcitabine activity was also evaluated in combination with Abraxane and Trematenib in both parental and resistant-clone cell lines. In addition, novel three-dimensional (3D) organoid cultures have been established from the two dimensional (2D) CRC cultures in order to verify the constancy of the model. Results: We established KRAS-mutant primary cell lines derived from patients' PA specimens. The cell lines' karyotype showed stability over multiple passages covering more than 6 months in continuous 2D culture. Five Abraxane resistant clones have been derived to date from two different parent cell lines and two gemcitabine resistant clones derived from one of these cell lines. The clones tested were 3 to 1000 times less sensitive to the drugs when compared to the parents. Notably, the Abraxane resistant clones also showed a greater resistance to Gemcitabine as compared to the parent line. In addition, the PA lines also showed an overall increasing sensitivity to Gemcitabine when pre- or co-treated with Trematenib. Molecular and genetic analyses are being performed to identify potential biomarkers of high therapeutic value. Conclusion: Taken together, the ease of culture, the genetic stability, the medium to high throughput ability to identify differences between patients sensitivity to FDA approved drugs, all confirm the power of this technology for on-demand in vitro use in PA research. Our approach now enables the high-resolution experiments necessary to better understand the underlying drug sensitivity and resistance mechanisms that directly affect clinical outcomes. Citation Format: Erika M. Parasido, Praathibha Sripadhan, Richard Schlegel, Michael J. Pishvaian, Jonathan Brody, Jordan Winter, Christopher Albanese. Patient-derived Pancreatic Adenocarcinoma cells: A new model system to define chemotherapy resistance mechanisms and to improve targeted personalized treatment. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B15.