Development and validation of a reversed-phase HPLC method for quantification of 1'-acetoxychavicol acetate content in a nanostructured lipid carrier formulation

Abstract 1'-acetoxychavicol acetate (ACA)-loaded nanostructured lipid carriers (NLCs) were formulated for prostate cancer therapy and to determine the optimal therapeutic dose, we developed a rapid, specific, and accurate reversed-phase high-performance liquid chromatography (RP-HPLC) method to quantify the ACA content in NLCs. The method was validated according to International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines. Chromatographic separation of ACA from the lipid components was performed with an Agilent 1220 Infinity LC system and ultraviolet detector using an Agilent Poroshell C18 column (4.6 x 250.0 mm). The mobile phase consisted of acetonitrile and water (80:20 [v/v]) with a flow rate of 0.8 mL/min in isocratic mode. Linearity of the standard curve was assessed at an ACA concentration range of 5-200 µg/mL, and a 1/x weighted linear regression was adopted for the calibration curve. The calculated limits of detection and quantification were 0.59 µg/mL and 1.79 µg/mL, respectively. The mean percent recovery of ACA was 100.02% (relative SD, 2%), and the coefficients of variation for intraday and interday assays were within the values required by the ICH. We also demonstrated robustness of the method by altering the mobile phase ratio and flow rate. Furthermore, we proved specificity of the method for ACA by comparing chromatograms of the blank NLC and ACA-NLC. Hence, we effectively used this validated method to determine the drug-loading capacity and entrapment efficiency of the NLCs.

Oncolytic HSV Therapy Modulates Vesicular Trafficking Inducing Cisplatin Sensitivity and Antitumor Immunity.

PURPOSE: Here we investigated the impact of oncolytic herpes simplex virus (HSV) treatment on cisplatin sensitivity of platinum-resistant ovarian cancer, and the impact of the combination on immunotherapy. EXPERIMENTAL DESIGN: Therapeutic efficacy of the combination was assessed in platinum-resistant human and murine ovarian cancer peritoneal metastatic mouse models (n = 9-10/group). RNA sequencing along with flow cytometry of splenocytes from treated mice was employed to examine the effect of antitumor immune response (n = 3/group). Anti-PD-1 antibody was performed to evaluate impact on checkpoint inhibition in vivo. RESULTS: Gene Ontology pathway analysis uncovered disruption of cellular extracellular vesicle (EV)-related pathways in infected cells (FDR = 2.97E-57). Mechanistically, we identified reduced expression of transporters expressed on EV implicated in cisplatin efflux. The increased cisplatin retention led to increased cisplatin-DNA adducts, which resulted in micronuclei and the subsequent activation of cGAS-STING pathway with a significant activation of innate immune cells and translated to an increase in antitumor immunity and efficacy. In mice bearing platinum-resistant ovarian cancer, we also observed a feedback induction of PD-L1 on tumor cells, which sensitized combination-treated mice to anti-PD-1 immune checkpoint therapy. CONCLUSIONS: To our knowledge, this is the first report to show HSV-induced cisplatin retention in infected cells. The consequential increased damaged DNA was then expelled from cells as micronuclei which resulted in induction of inflammatory responses and education of antitumor immunity. The combination therapy also created an environment that sensitized tumors to immune checkpoint therapy.

Protein expression profiling identifies differential modulation of homologous recombination by platinum-based antitumor agents.

PURPOSE: Oxaliplatin and satraplatin demonstrate activity against cisplatin-resistant tumor cells. Although the two platinum analogs are structurally-related, oxaliplatin is more active. Therefore, studies focusing on protein expression profiling were undertaken to identify the molecular mechanism for the difference in antitumor activity. METHODS: We included cisplatin as reference and DAP as a Pt(IV)-prodrug of oxaliplatin to offset Pt(IV) status of satraplatin, and utilized A2780, cisplatin-resistant 2780CP/Cl-16, U2OS, and HCT-116 tumor cells in the investigation. Protein expressions following drug exposures were examined by reverse-phase protein array and ingenuity pathway analysis. Cell cycle was assessed by flow cytometry, cytotoxicity by growth inhibition assay, and homologous recombination (HR) by a GFP reporter assay. RESULTS: Clustering analysis paired oxaliplatin with DAP and, surprisingly, satraplatin with cisplatin. This correlated with differential upregulation of p53/p21 pathway, with S and G2/M arrests by cisplatin and satraplatin in contrast to G1 arrest by oxaliplatin and DAP. Moreover, Rad51 and BRCA1 were severely downregulated by oxaliplatin and DAP, but not cisplatin and satraplatin. As a result, HR was inhibited only by oxaliplatin and DAP and this also contributed to their greater drug activity over cisplatin and satraplatin. CONCLUSIONS: Oxaliplatin and DAP robustly activate p53 and p21, which downregulate HR proteins to enhance drug activity. More significantly, since oxaliplatin induces a BRCAness state, it may have potential against BRCA-proficient cancers. Satraplatin, on the other hand, resembled cisplatin in its protein expression profile, which indicates that small changes in chemical structure can substantially alter signal transduction pathways to modulate drug activity.

Oxaliplatin Pt(IV) prodrugs conjugated to gadolinium-texaphyrin as potential antitumor agents.

Described here is the development of gadolinium(III) texaphyrin-platinum(IV) conjugates capable of overcoming platinum resistance by 1) localizing to solid tumors, 2) promoting enhanced cancer cell uptake, and 3) reactivating p53 in platinum-resistant models. Side by side comparative studies of these Pt(IV) conjugates to clinically approved platinum(II) agents and previously reported platinum(II)-texaphyrin conjugates demonstrate that the present Pt(IV) conjugates are more stable against hydrolysis and nucleophilic attack. Moreover, they display high potent antiproliferative activity in vitro against human and mouse cell cancer lines. Relative to the current platinum clinical standard of care (SOC), a lead Gd(III) texaphyrin-Pt(IV) prodrug conjugate emerging from this development effort was found to be more efficacious in subcutaneous (s.c.) mouse models involving both cell-derived xenografts and platinum-resistant patient-derived xenografts. Comparative pathology studies in mice treated with equimolar doses of the lead Gd texaphyrin-Pt(IV) conjugate or the US Food and Drug Administration (FDA)-approved agent oxaliplatin revealed that the conjugate was better tolerated. Specifically, the lead could be dosed at more than three times (i.e., 70 mg/kg per dose) the tolerable dose of oxaliplatin (i.e., 4 to 6 mg/kg per dose depending on the animal model) with little to no observable adverse effects. A combination of tumor localization, redox cycling, and reversible protein binding is invoked to explain the relatively increased tolerability and enhanced anticancer activity seen in vivo. On the basis of the present studies, we conclude that metallotexaphyrin-Pt conjugates may have substantial clinical potential as antitumor agents.

Cisplatin in Combination with MDM2 Inhibition Downregulates Rad51 Recombinase in a Bimodal Manner to Inhibit Homologous Recombination and Augment Tumor Cell Kill.

Dysfunction of p53 and resistance to cancer drugs can arise through mutually exclusive overexpression of MDM2 or MDM4. Cisplatin-resistant cells, however, can demonstrate increased binding of both MDM2 and MDM4 to p53 but in absence of cellular overexpression. Whether MDM2 inhibitors alone can activate p53 in these resistant cells was investigated with the goal to establish the mechanism for potential synergy with cisplatin. Thus, growth inhibition by individual drugs and combinations was assessed by a colorimetric assay. Drug-treated parental A2780 and resistant tumor cells were also examined for protein expression using immunoblot and reverse phase protein array (RPPA) and then subjected to Ingenuity Pathway Analysis (IPA). Gene expression was assessed by real-time polymerase chain reaction, DNA damage by confocal microscopy, cell cycle by flow cytometry, and homologous recombination (HR) by a GFP reporter assay. Our results demonstrate that Nutlin-3 but not RITA (reactivation of p53 and induction of tumor cell apoptosis) effectively disrupted the p53-MDM2-MDM4 complex to activate p53, which increased robustly with cisplatin/Nutlin-3 combination and enhanced antitumor effects more than either agent alone. RPPA, IPA, and confocal microscopy provided evidence for an "apparent" increase in DNA damage resulting from HR inhibition by cisplatin/Nutlin-3. Molecularly, the specific HR protein Rad51 was severely downregulated by the combination via two mechanisms: p53-dependent transrepression and p53/MDM2-mediated proteasomal degradation. In conclusion, Nutlin-3 fully destabilizes the p53-MDM2-MDM4 complex and synergizes with cisplatin to intensify p53 function, which then downregulates Rad51 through a bimodal mechanism. As a result, HR is inhibited and antitumor activity enhanced in otherwise HR-proficient sensitive and resistant tumor cells. SIGNIFICANCE STATEMENT: Rad51 downregulation by the combination of cisplatin and Nutlin-3 inhibits homologous recombination (HR), which leads to persistence in DNA damage but not an increase. Thus, inhibition of HR enhances antitumor activity in otherwise HR-proficient sensitive and resistant tumor cells.

Platinum(IV)-Ferrocene Conjugates and Their Cyclodextrin Host-Guest Complexes.

Reported here are new platinum(IV) (Pt(IV)) complexes bearing ferrocene (Fc) moieties. These systems differ from one another only by the nature of the functional group (ester vs amide) connecting the linker to the Fc subunits. This minor structural variation (one atom difference) leads to major differences in solubility, stability, and antiproliferative activity against lung (A549) cancer cells. The host-guest chemistry of these complexes was investigated in an aqueous medium in the presence of ß-cyclodextrins (ß-CD), either free or in the form of a covalently linked Fc-Pt-ß-CD hybrid. An inclusion complex between Fc and ß-CD is formed in aqueous media, presumably as a result of hydrophobic interactions involving the Fc and the inner ß-CD cavity. Consequently, it proved possible to use a ß-CD-based strategy to purify the Pt-Fc conjugates in this study under aqueous conditions (by means of C18 silica gel columns). The use of a ß-CD adjuvant also allowed dimethyl sulfoxide (DMSO) to be avoided as an organic cosolvent in cell studies. The amide version reported here (2) proved to be more soluble, more stable, and more active than the ester analogue (11) in A549 cells. The use of a ß-CD functionalized with a fluorescent probe allowed intracellular Pt-Fc localization to be visualized by confocal fluorescence microscopy.

PTGER3 induces ovary tumorigenesis and confers resistance to cisplatin therapy through up-regulation Ras-MAPK/Erk-ETS1-ELK1/CFTR1 axis.

BACKGROUND: Inflammatory mediator prostaglandin E2-prostaglandin E2 receptor EP3 (PTGER3) signaling is critical for tumor-associated angiogenesis, tumor growth, and chemoresistance. However, the mechanism underlying these effects in ovarian cancer is not known. METHODS: An association between higher tumoral expression of PTGER3 and shorter patient survival in the ovarian cancer dataset of The Cancer Genome Atlas prompted investigation of the antitumor effects of PTGER3 downmodulation. PTGER3 mRNA and protein levels were higher in cisplatin-resistant ovarian cancer cells than in their cisplatin-sensitive counterparts. FINDINGS: Silencing of PTGER3 via siRNA in cancer cells was associated with decreased cell growth and less invasiveness, as well as cell-cycle arrest and increased apoptosis, mediated through the Ras-MAPK/Erk-ETS1-ELK1/CFTR1 axis. Furthermore, sustained PTGER3 silencing with multistage vector and liposomal 2'-F-phosphorodithioate-siRNA-mediated silencing of PTGER3 combined with cisplatin resulted in robust antitumor effects in cisplatin-resistant ovarian cancer models. INTERPRETATION: These findings identify PTGER3 as a potential therapeutic target in chemoresistant ovarian cancers expressing high levels of this oncogenic protein. FUND: National Institutes of Health/National Cancer Institute, USA.

Overview of the oncogenic signaling pathways in colorectal cancer: Mechanistic insights.

Colorectal cancer is a multifaceted disease which is therapeutically challenging. Based on insights gleaned from almost a quarter century of research, it is obvious that deregulation of spatio-temporally controlled signaling pathways play instrumental role in development and progression of colorectal cancer. High-throughput technologies have helped to develop a sharper and broader understanding of the wide ranging signal transduction cascades which also contribute to development of drug resistance, loss of apoptosis and, ultimately, of metastasis. In this review, we have set the spotlight on role of JAK/STAT, TGF/SMAD, Notch, WNT/ß-Catenin, SHH/GLI and p53 pathways in the development and progression of colorectal cancer. We have also highlighted recent reports on TRAIL-mediated pathways and molecularly distinct voltage-gated sodium channels in colorectal cancer.

Curcumin-Free Turmeric Exhibits Activity against Human HCT-116 Colon Tumor Xenograft: Comparison with Curcumin and Whole Turmeric.

Extensive research within last two decades has indicated that curcumin extracted from turmeric (Curcuma longa), exhibits anticancer potential, in part through the modulation of inflammatory pathways. However, the residual antitumor activity of curcumin-free turmeric (CFT) relative to curcumin or turmeric is not well-understood. In the present study, therefore, we determined activities of these agents in both in vitro and in vivo models of human HCT-116 colorectal cancer (CRC). When examined in an in vitro antiproliferative, clonogenic or anti-inflammatory assay system, we found that curcumin was highly active whereas turmeric and CFT had relatively poor activity against CRC cells. However, when examined in vivo at an oral dose of either 100 or 500 mg/kg given to nude mice bearing CRC xenografts, all three preparations of curcumin, turmeric, and CFT similarly suppressed the growth of the xenograft. The effect of CFT on suppression of tumor growth was dose-dependent, with 500 mg/kg tending to be more effective than 100 mg/kg. Interestingly, 100 mg/kg curcumin or turmeric was found to be more effective than 500 mg/kg. When examined in vivo for the expression of biomarkers associated with cell survival (cIAP-1, Bcl-2, and survivin), proliferation (Ki-67 and cyclin D1) and metastasis (ICAM-1 and VEGF), all were down-modulated. These agents also suppressed inflammatory transcription factors (NF-κB and STAT3) in tumor cells. Overall, our results with CFT provide evidence that turmeric must contain additional bioactive compounds other than curcumin that, in contrast to curcumin, exhibit greater anticancer potential in vivo than in vitro against human CRC. Moreover, our study highlights the fact that the beneficial effects of turmeric and curcumin in humans may be more effectively realized at lower doses, whereas CFT could be given at higher doses without loss in favorable activity.

Drug-dependent functionalization of wild-type and mutant p53 in cisplatin-resistant human ovarian tumor cells.

Cisplatin (cis-Pt) resistance in tumor cells from p53 dysfunction is a significant clinical problem. Although mutation can inhibit p53 function, >60% of p53 mutants retain normal function according to literature reports. Therefore, we examined the status of p53 in cisplatin-resistant ovarian tumor models and its functional response to cis-Pt and the mechanistically-distinct non-cross-resistant oxaliplatin (oxali-Pt). Relative to sensitive A2780 cells harboring wild-type p53, the 2780CP/Cl-16, OVCAR-10, Hey and OVCA-433 cell lines were 10- to 30-fold resistant to cis-Pt, but was substantially circumvented by oxali-Pt. Mutant p53 in 2780CP/Cl-16 (p53V172F) and OVCAR-10 (p53V172F and p53G266R) cells, predicted as non-functional in p53 database, displayed attenuated response to cis-Pt, as did the polymorphic p53P72R (functionally equivalent to wild-type p53) in HEY and OVCA-433 cell lines. However, p53 was robustly activated by oxali-Pt in all cell lines, with resultant drug potency confirmed as p53-dependent by p53 knockout using CRISPR/Cas9 system. This p53 activation by oxali-Pt was associated with phosphorylation at Ser20 by MEK1/2 based on inhibitor and kinase studies. Cis-Pt, however, failed to phosphorylate Ser20 due to downregulated Chk2, and its clinical impact validated by reduced overall survival of ovarian cancer patients according to TCGA database. In conclusion, cis-Pt resistance occurs in both wild-type and mutant p53 ovarian cancer cells, but is associated with loss of Ser20 phosphorylation. However, these mutant p53, like polymorphic p53, are functional and activated by oxali-Pt-induced Ser20 phosphorylation. Thus, the potential exists for repurposing oxali-Pt or similar drugs against refractory cancers harboring wild-type or specific mutant p53.

Functional Activation of Mutant p53 by Platinum Analogues in Cisplatin-Resistant Cells Is Dependent on Phosphorylation.

Dysfunctionality of the p53 tumor suppressor is a major cause of therapeutic drug resistance in cancer. Recently, we reported that mutant, but otherwise functional, p53v172F was inactivated in cisplatin-resistant 2780CP/Cl-16 and 2780CP/Cl-24 human ovarian tumor cells by increased recruitment of the inhibitor MDM4. The current study demonstrates that, unlike cisplatin, platinum analogues oxaliplatin and DACH-diacetato-dichloro-Pt(IV) (DAP) strongly stabilize and activate p53v172F in resistant cells, as indicated by prolonged p53 half-life and transactivation of targets p21 (CDKN1A) and MDM2. This increase in MDM2 reduced MDM4 levels in cell lysates as well as the p53 immunocomplex and prevented reversion of p53 to the inactive p53-MDM2-MDM4-bound state. Phosphorylation of p53 at Ser15 was demonstrated by all three drugs in sensitive A2780 and corresponding resistant 2780CP/Cl-16 and 2780CP/Cl-24 cell lines. However, cisplatin induced Ser20 phosphorylation in A2780 cells only, but not in resistant cells; in contrast, both DAP and oxaliplatin induced this phosphorylation in all three cell lines. The inference that Ser20 phosphorylation is more important for p53 activation was confirmed by ectopic expression of a phosphomimetic (S20D) mutant p53 that displayed reduced binding, relative to wild-type p53, to both MDM2 and MDM4 in p53-knockout A2780 cells. In consonance, temporal studies demonstrated drug-induced Ser15 phosphorylation coincided with p53 stabilization, whereas Ser20 phosphorylation coincided with p53 transactivation.Implications: Cisplatin fails to activate the pathway involved in phosphorylating mutant p53v172F at Ser20 in resistant cells, but this phosphorylation is restored by oxaliplatin and DAP that reactivates p53 function and circumvents cisplatin resistance. Mol Cancer Res; 15(3); 328-39. ©2016 AACR.

New bipyridine gold(III) dithiocarbamate-containing complexes exerted a potent anticancer activity against cisplatin-resistant cancer cells independent of p53 status.

We synthesized, characterized and tested in a panel of cancer cell lines, nine new bipyridine gold(III) dithiocarbamate-containing complexes. In vitro studies demonstrated that compounds 1, 2, 4, 5, 7 and 8 were the most cytotoxic in prostate, breast, ovarian cancer cell lines and in Hodgkin lymphoma cells with IC50 values lower than the reference drug cisplatin. The most active compound 1 was more active than cisplatin in ovarian (A2780cis and 2780CP-16) and breast cancer cisplatin-resistant cells. Compound 1 determined an alteration of the cellular redox homeostasis leading to increased ROS levels, a decrease in the mitochondrial membrane potential, cytochrome-c release from the mitochondria and activation of caspases 9 and 3. The ROS scavenger NAC suppressed ROS generation and rescued cells from damage. Compound 1 resulted more active in tumor cells than in normal human Mesenchymal stromal cells. Gold compounds were active independent of p53 status: exerted cytotoxic effects on a panel of non-small cell lung cancer cell lines with different p53 status and in the ovarian A2780 model where the p53 was knocked out. In conclusion, these promising results strongly indicate the need for further preclinical evaluation to test the clinical potential of these new gold(III) complexes.

Activation of Platinum(IV) Prodrugs By Motexafin Gadolinium as a Redox Mediator.

Water-soluble platinum(IV) prodrugs, which proved kinetically stable to reduction in the presence of physiological concentration of ascorbate, were quickly reduced to their active form, oxaliplatin, when co-incubated with a macrocycle metallotexaphyrin (i.e., Motexafin Gadolinium (MGd)). The reduction of Pt(IV) to Pt(II) promoted by MGd occurs in cell culture as well, leading to an increase in the antiproliferative activity of the Pt(IV) species in question. The mediated effect is proportional to the concentration of MGd and gives rise to an enhancement when the prodrug is relatively hydrophilic. MGd is known to localize/accumulate preferentially in tumor tissues. Thus, the present "activation by reduction" approach may allow for the cancer-selective enhancement in the cytotoxicity of Pt(IV) prodrugs.

Platelet-derived growth factor (PDGF) signalling in cancer: rapidly emerging signalling landscape.

Platelet-derived growth factor (PDGF)-mediated signalling has emerged as one of the most extensively and deeply studied biological mechanism reported to be involved in regulation of growth and survival of different cell types. However, overwhelmingly increasing scientific evidence is also emphasizing on dysregulation of spatio-temporally controlled PDGF-induced signalling as a basis for cancer development. We partition this multi-component review into recently developing understanding of dysregulation PDGF signalling in different cancers, how PDGF receptors are quantitatively controlled by microRNAs. Moreover, we also summarize most recent advancements in therapeutic targeting of PDGFR as evidenced by preclinical studies. Better understanding of the PDGF-induced intracellular signalling in different cancers will be helpful in catalysing the transition from a segmented view of cancer biology to a conceptual continuum.

Phase I study of azacitidine and oxaliplatin in patients with advanced cancers that have relapsed or are refractory to any platinum therapy.

BACKGROUND: Demethylation process is necessary for the expression of various factors involved in chemotherapy cytotoxicity or resistance. Platinum-resistant cells may have reduced expression of the copper/platinum transporter CTR1. We hypothesized that azacitidine and oxaliplatin combination therapy may restore platinum sensitivity. We treated patients with cancer relapsed/refractory to any platinum compounds (3 + 3 study design) with azacitidine (20 to 50 mg/m(2)/day intravenously (IV) over 15 to 30 min, D1 to 5) and oxaliplatin (15 to 30 mg/m(2)/day, IV over 2 h, D2 to 5) (maximum, six cycles). Platinum content, LINE1 methylation (surrogate of global DNA methylation), and CTR1 expression changes (pre- vs. post-treatment) were assessed. Drug pharmacokinetics were analyzed. RESULTS: Thirty-seven patients were treated. No dose-limiting toxicity (DLT) was noted at the maximum dose. The most common adverse events were anemia and fatigue. Two (5.4%) patients had stable disease and completed six cycles of therapy. Oxaliplatin (D2) and azacitidine (D1 and 5) mean systemic exposure based on plasma AUCall showed dose-dependent interaction whereby increasing the dose of oxaliplatin reduced the mean azacitidine exposure and vice versa; however, no significant differences in other non-compartmental modeled parameters were observed. Blood samples showed universal reduction in global DNA methylation. In tumor samples, hypomethylation was only observed in four out of seven patients. No correlation between blood and tumor demethylation was seen. The mean cytoplasmic CTR1 score decreased. The pre-dose tumor oxaliplatin levels ranged from <0.25 to 5.8 µg/g tumor. The platinum concentration increased 3- to 18-fold. No correlation was found between CTR1 score and oxaliplatin level, which was found to have a trend toward correlation with progression-free survival. CONCLUSIONS: Oxaliplatin and azacitidine combination therapy was safe. CTR1 expression was not correlated with methylation status or tissue platinum concentration.

Photoinduced reduction of Pt(IV) within an anti-proliferative Pt(IV)-texaphyrin conjugate.

In an effort to increase the stability and control the platinum reactivity of platinum-texaphyrin conjugates, two Pt(IV) conjugates were designed, synthesized, and studied for their ability to form DNA adducts. They were also tested for their anti-proliferative effects using wild-type and platinum-resistant human ovarian cancer cell lines (A2780 and 2780CP, respectively). In comparison to an analogous first-generation Pt(II) chimera, one of the new conjugates provided increased stability in aqueous environments. Using a combination of (1) H NMR spectroscopy and FAAS (flameless atomic-absorption spectrometry), it was found that the Pt(IV) center within this conjugate undergoes photoinduced reduction to Pt(II) upon exposure to glass-filtered daylight, resulting in an entity that binds DNA in a controlled manner. Under conditions in which the Pt(IV) complex is reduced to the corresponding Pt(II) species, these new conjugates demonstrated potent anti-proliferative activity in both test ovarian cancer cell lines.