Gambogic Acid Sensitizes Ovarian Cancer Cells to Doxorubicin Through ROS-Mediated Apoptosis

Ovarian cancer is one human malignancy which has response portly to doxorubicin. The anti-cancer activity of gambogic acid has been tested in in vitro and in vivo studies. In this study, we showed that gambogic acid, a natural compound, could potentiate the anticancer activity of doxorubicin in ovarian cancer through ROS-mediated apoptosis. Platinum-resistant human ovarian cancer cell line (SKOV-3) was treated with gambogic acid, doxorubicin, or the combination of both to investigate cell proliferation and apoptosis. We found that the combination of gambogic acid and doxorubicin causes synergistic loss of cell viability in SKOV-3 cells and this synergistic effect correlated with increased cellular ROS accumulation. Moreover, in vivo results showed that gambogic acid and doxorubicin combination resulted in a synergistic suppressing effect on tumor growth in ovarian cancer mice model. Taken together, the results suggested that doxorubicin in combination with gambogic acid might provide a promising therapeutic strategy to enhance chemosensitivity of ovarian cancer to doxorubicin.     

Membrane-rigidifying effects of anti-cancer dietary factors

Since several anti-cancer drugs interact with cell membrane lipids, the effects of anti-cancer dietary factors on liposomal membranes with different lipid composition were comparatively studied by measuring fluorescence polarization. Fluidity was imparted on both hydrophobic and hydrophilic regions of lipid bilayers by decreasing cholesterol and increasing unsaturated phosphatidylcholine in membranes. At 0.625-10 microM, (-)-epigallocatechin gallate, genistein, apigenin, resveratrol and a reference anti-cancer drug, doxorubicin, rigidified the tumor cell model membranes consisting of 20 mol% cholesterol and 80 mol% phosphatidylcholine with the acyl chain 18:1/16:0 ratio of 1.0, but not daidzein. They were more effective on the membrane core than the membrane surface. Quercetin showed a biphasic effect on the hydrophobic regions of membrane lipid bilayers to rigidify above 5 microM and fluidize below 2.5 microM. In contrast, anti-cancer dietary factors and doxorubicin were not or much less effective in rigidifying the normal cell model membranes consisting of 40 mol% cholesterol and 60 mol% phosphatidylcholine with the acyl chain 18:1/16:0 ratio of 0.5. The membrane-rigidifying effects were greater depending on a decrease of the cholesterol/phosphatidylcholine ratio and an increase of the phosphatidylcholine unsaturation degree. Membrane-active dietary factors and doxorubicin inhibited the growth of mouse myeloma cells at 10-100 microM, while the growth inhibition by membrane-inactive daidzein was relatively weak. Anti-cancer dietary factors appear to act on more fluid membranes like tumor cells as well as doxorubicin to induce rigidification, especially in the hydrocarbon core of membrane lipids, which is determined by the composition of cholesterol and unsaturated phospholipids.     

Simulation model of doxorubicin activity in islets of human breast cancer cells

During cytotoxic chemotherapy, cancer cells are exposed to a dynamic concentration-versus-time curve. Besides the area under this curve, the shape of this curve may determine the cytotoxic effect. This report describes the concept that cell damage is determined by the molar drug accumulation history inside the tumor cells. Cell numbers of large populations of human MCF-7 cells exposed to three different doxorubicin concentration-versus-time profiles were recorded for 31 days. The drug accumulation history in the cells was calculated using cellular drug transport parameters derived from doxorubicin uptake and efflux measurements on MCF-7 cells attached to culture dishes. Recovery of the proliferation rate of a cell population after drug exposure was described using a mathematical model of cell damage. The model fitted well to the proliferation assays. It allowed for comparison of the effects of changes in doxorubicin concentration-versus-time profiles in vitro. The model was then used to predict the effect of the changes in the doxorubicin concentration profile in vivo, in tumor islets, after a bolus injection of doxorubicin. In the model doxorubicin exposure resulted in less cell damage inside the tumor islets than at the rim.     

Circadian chemotherapy for gynecological and genitourinary cancers

The circadian timing of surgery, anticancer drugs, radiation therapy, and biologic agents can result in improved toxicity profiles, tumor control, and host survival. Optimally timed cancer chemotherapy with doxorubicin or pirarubicin (06:00h) and cisplatin (18:00h) enhanced the control of advanced ovarian cancer while minimizing side effects, and increased the response rate in metastatic endometrial cancer. Therapy of metastatic bladder cancer with doxorubicin–cisplatin was made more tolerable by this same circadian approach resulting in a 57% objective response rate. This optimally timed therapy is also effective in the adjuvant setting, decreasing the expected frequency of metastasis from locally advanced bladder cancer. Circadian fluorodeoxyuridine (FUDR) continuous infusion (70% of the daily dose given between 15:00h and 21:00h) has been shown effective for metastatic renal cell carcinoma resulting in 29% objective response and stable disease of more than 1 yr duration in the majority of patients. Toxicity is reduced markedly when FUDR infusion is modulated to circadian rhythms. In a multicenter trial in patients with metastatic renal cell cancer, patients were randomized to a flat or a circadian-modified FUDR infusion. This study confirmed a significant difference in toxicity and dose intensity, favoring the circadian-modified group. Hormone refractory metastatic prostate cancer has been treated with circadian-timed FUDR chemotherapy; however, without objective response. Biological agents such as interferon-α and IL-2 have shown low but effective disease control in metastatic renal cell cancer, however, with much toxicity. Each of these cytokines shows circadian stage dependent toxicity and efficacy in model systems. In summary, the timing of anthracycline, platinum, and fluoropyrimidine-based drug therapies during the 24h is relevant to the toxic–therapeutic ratio of these agents in the treatment of gynecologic and genitourinary cancers.     

Ras puts the brake on doxorubicin-mediated cell death in p53-expressing cells

Doxorubicin is one of the most effective molecules used in the treatment of various tumors. Contradictory reports often open windows to understand the role of p53 tumor suppressor in doxorubicin-mediated cell death. In this report, we provide evidences that doxorubicin induced more cell death in p53-negative tumor cells. Several cells, having p53 basal expression, showed increase in p53 DNA binding upon doxorubicin treatment. Doxorubicin induced cell death in p53-positive cells through expression of p53-dependent genes and activation of caspases and caspase-mediated cleavage of cellular proteins. Surprisingly, in p53-negative cells, doxorubicin-mediated cell death was more aggressive (faster and intense). Doxorubicin increased the amount of Fas ligand (FasL) by enhancing activator protein (AP) 1 DNA binding in both p53-positive and p53-negative cells, but the basal expression of Fas was higher in p53-negative cells. Anti-FasL antibody considerably protected doxorubicin-mediated cell death in both types of cells. Activation of caspases was faster in p53-negative cells upon doxorubicin treatment. In contrast, the basal expression of Ras oncoprotein was higher in p53-positive cells, which might increase the basal expression of Fas in these cells. Overexpression of Ras decreased the amount of Fas in p53-negative cells, thereby decreasing doxorubicin-mediated aggressive cell death. Overall, this study will help to understand the much studied chemotherapeutic drug, doxorubicin-mediated cell signaling cascade, that leads to cell death in p53-positive and -negative cells. High basal expression of Fas might be an important determinant in doxorubicin-mediated cell death in p53-negative cells.     

A mechanical model of tumor encapsulation and transcapsular spread

In this paper, a mathematical modeling framework is presented which describes the growth, encapsulation, and transcapsular spread of solid tumors. The model is based on the physical forces and cellular interactions involved in tumorigenesis and is used to test and compare the active (foreign body hypothesis) and passive (expansive growth hypothesis) hypotheses of capsule formation, such investigations being ideally suited to our mechanical model. The model simulations lead us to predict that, although an active response can successfully control tumor growth via the deposition of large amounts of collagen, this alone is insufficient for capsule formation. In contrast, a solely passive responsive is capable of producing an encapsulated tumor with minimal accumulation of connective tissue within the tumor. When both responses are active, a denser capsule forms and there is a significant increase in connective tissue within the tumor. Using a modified version of the model, in which tumor cells are assumed to produce degradative proteases at a rate which depends on the pressure they experience, it is also possible to show that transcapsular spread or invasion of the tumor may be due to the production by the tumor cells of proteases and their subsequent action.     

Potentiating effects of GHRH analogs on the response to chemotherapy

Growth hormone releasing hormone (GHRH) from hypothalamus nominatively stimulates growth hormone release from adenohypophysis. GHRH is also produced by cancers, acting as an autocrine/paracrine growth factor. This growth factor function is seen in lymphoma, melanoma, colorectal, liver, lung, breast, prostate, kidney, bladder cancers. Pituitary type GHRH receptors and their splice variants are also expressed in these malignancies. Synthetic antagonists of the GHRH receptor inhibit proliferation of cancers. Besides direct inhibitory effects on tumors, GHRH antagonists also enhance cytotoxic chemotherapy. GHRH antagonists potentiate docetaxel effects on growth of H460 non-small cell lung cancer (NSCLC) and MX-1 breast cancer plus suppressive action of doxorubicin on MX-1 and HCC1806 breast cancer. We investigated mechanisms of antagonists on tumor growth, inflammatory signaling, doxorubicin response, expression of drug resistance genes, and efflux pump function. Triple negative breast cancer cell xenografted into nude mice were treated with GHRH antagonist, doxorubicin, or their combination. The combination reduced tumor growth, inflammatory gene expression, drug-resistance gene expression, cancer stem-cell marker expression, and efflux-pump function. Thus, antagonists increased the efficacy of doxorubicin in HCC1806 and MX-1 tumors. Growth inhibition of H460 NSCLC by GHRH antagonists induced marked downregulation in expression of prosurvival proteins K-Ras, COX-2, and pAKT. In HT-29, HCT-116 and HCT-15 colorectal cancer lines, GHRH antagonist treatment caused cellular arrest in S-phase of cell cycle, potentiated inhibition of in vitro proliferation and in vivo growth produced by S-phase specific cytotoxic agents, 5-FU, irinotecan and cisplatin. This enhancement of cytotoxic therapy by GHRH antagonists should have clinical applications.     

Slowdown of growth controls cellular differentiation

How can changes in growth rate affect the regulatory networks behavior and the outcomes of cellular differentiation? We address this question by focusing on starvation response in sporulating Bacillus subtilis. We show that the activity of sporulation master regulator Spo0A increases with decreasing cellular growth rate. Using a mathematical model of the phosphorelay—the network controlling Spo0A—we predict that this increase in Spo0A activity can be explained by the phosphorelay protein accumulation and lengthening of the period between chromosomal replication events caused by growth slowdown. As a result, only cells growing slower than a certain rate reach threshold Spo0A activity necessary for sporulation. This growth threshold model accurately predicts cell fates and explains the distribution of sporulation deferral times. We confirm our predictions experimentally and show that the concentration rather than activity of phosphorelay proteins is affected by the growth slowdown. We conclude that sensing the growth rates enables cells to indirectly detect starvation without the need for evaluating specific stress signals.     

Enhanced transformation and chemosensitivity of NIH3T3 cells transduced with hepatoma up-regulated protein

Hepatoma up-regulated protein (HURP) is a recently identified novel cell-cycle-regulated gene. The HURP gene is overexpressed in human hepatocellular carcinoma and transitional cell carcinoma. The cellular function of HURP is not fully understood. In this study, the NIH3T3 cells transduced with the exogenous HURP gene manifested the general characteristics of tumor cells, which had higher growth rate in low-serum media and advanced ability of colony formation on agarose-based plates. Transduced HURP was capable of specifically enhancing the chemosensitivity of deoxycytosine analogs, such as gemcitabine, ARA-C, and 5-AZA-CdR, but neither had an effect on the response of DNA intercalating agents, such as cisplatin, carboplatin, and doxorubicin, nor on the response of microtubule stabilizers, such as paclitaxel, docetaxel, and vinblastine. These results indicate that the HURP gene might be a potential oncogenic gene and capable of enhancing the chemosensitivity of deoxycytosine analogs in NIH3T3 cells.     

Successful treatment of advanced murine renal cell cancer by bicompartmental adoptive chemoimmunotherapy

The most challenging aspect of cancer treatment remains the management of invasive and metastatic tumor growth. Recent progress in the development and use of biologic response modifiers for immunomodulation has raised the possibility that the immune system can be used as an additional antitumor treatment modality in conjunction with surgery, chemotherapy, and/or radiotherapy for the treatment of established tumors and their metastases. As a model for adoptive chemoimmunotherapy (ACIT) of renal cancer we have used a murine renal cancer (Renca) of spontaneous origin that mimics the tumor progression characteristically observed for human renal cell carcinoma. In the present study, we demonstrate that broadly cytotoxic lymphocytes, generated by in vitro culture with human recombinant interleukin 2, and used in conjunction with the chemotherapeutic drug doxorubicin hydrochloride, are effective in treating invasive and metastatic renal cell cancer. Administration of ACIT i.v. or i.p., alone, or after nephrectomy of the tumor-bearing kidney, did not cure mice with stage II (locoregional invasive tumor) or stage III (lymph node metastases) disease. In contrast, nephrectomy followed by simultaneous bicompartmental i.v. and i.p. ACIT administration cured 80% of mice with either stage II or stage III Renca. These data demonstrate that simultaneous bicompartmental ACIT affords dramatically improved cure rates for advanced and metastatic Renca. This effect most likely results from efficient control of both locoregional and metastatic tumor growth.     

Predictive Modeling of Neuroblastoma Growth Dynamics in Xenograft Model After Bevacizumab Anti-VEGF Therapy

Neuroblastoma is the leading cause of cancer death in young children. Although treatment for neuroblastoma has improved, the 5-year survival rate of patients still remains less than half. Recent studies have indicated that bevacizumab, an anti-VEGF drug used in treatment of several other cancer types, may be effective for treating neuroblastoma as well. However, its effect on neuroblastoma has not been well characterized. While traditional experiments are costly and time-consuming, mathematical models are capable of simulating complex systems quickly and inexpensively. In this study, we present a model of vascular tumor growth of neuroblastoma IMR-32 that is complex enough to replicate experimental data across a range of tumor cell properties measured in a suite of in vitro and in vivo experiments. The model provides quantitative insight into tumor vasculature, predicting a linear relationship between vasculature and tumor volume. The tumor growth model was coupled with known pharmacokinetics and pharmacodynamics of the VEGF blocker bevacizumab to study its effect on neuroblastoma growth dynamics. The results of our model suggest that total administered bevacizumab concentration per week, as opposed to dosage regimen, is the major determining factor in tumor suppression. Our model also establishes an exponentially decreasing relationship between administered bevacizumab concentration and tumor growth rate.     

Community dynamics, trade-offs, invasion criteria and the evolution of host resistance to microparasites

A hybrid cellular automaton model is described and used to simulate early tumor growth and examine the roles of host tissue vascular density and tumor metabolism in the ability of a small number of monoclonal transformed cells to develop into an invasive tumor. The model incorporates normal cells, tumor cells, necrotic or empty space, and a random network of native microvessels as components of a cellular automaton state vector. Diffusion of glucose and H(+)ions (the latter largely resulting from the tumor's excessive reliance on anaerobic metabolism) to and from the microvessels, and their utilization or production by cells, is modeled through the solution of differential equations. In this way, the cells and microvessels affect the extracellular concentrations of glucose and H(+)which, in turn, affect the evolution of the automaton. Simulations of the model demonstrate that: (i) high tumor H(+)ion production is favorable for tumor growth and invasion; however for every H(+)ion production rate, there exists a range of optimal microvessel densities (leading to a local pH favorable to tumor but not to normal cells) for which growth and invasion is most effective, (ii) at vascular densities below this range, both tumor and normal cells die due to excessively low pH, (iii) for vascular densities above the optimal range the microvessel network is highly efficient at removing acid and therefore the tumor cells lose their advantage over normal cells gained by high local H(+)concentration. While significant spatial gradients of glucose formed, no regions of detrimentally poor glucose perfusion (for either cell type) were observed, regardless of microvessel density. Depending on metabolic phenotype, a variety of tumor morphologies similar to those clinically observed were realized in the simulations. Lastly, a sharp transition (analogous to that of the adenoma-carcinoma sequence) between states of initial tumor confinement and efficient invasiveness was observed when H(+)production reached a critical value.     

A Microenvironment Based Model of Antimitotic Therapy of Gompertzian Tumor Growth

A model of tumor growth, based on two-compartment cell population dynamics, and an overall Gompertzian growth has been previously developed. The main feature of the model is an inter-compartmental transfer function that describes the net exchange between proliferating (P) and quiescent (Q) cells and yields Gompertzian growth for tumor cell population N = P + Q. Model parameters provide for cell reproduction and cell death. This model is further developed here and modified to simulate antimitotic therapy. Therapy decreases the reproduction-rate constant and increases the death-rate constant of proliferating cells with no direct effect on quiescent cells. The model results in a system of two ODE equations (in N and P/N) that has an analytical solution. Net tumor growth depends on support from the microenvironment. Indirectly, this is manifested in the transfer function, which depends on the proliferation ratio, P/N. Antimitotic therapy will change P/N, and the tumor responds by slowing the transfer rate from P to Q. While the cellular effects of therapy are modeled as dependent only on antimitotic activity of the drug, the tumor response also depends on the tumor age and any previous therapies—after therapy, it is not the same tumor. The strength of therapy is simulated by the parameter λ, which is the ratio of therapy induced net proliferation rate constant versus the original. A pharmacodynamic factor inversely proportional to tumor size is implemented. Various chemotherapy regimens are simulated and the outcomes of therapy administered at different time points in the life history of the tumor are explored. Our analysis shows: (1) for a constant total dose administered, a decreasing dose schedule is marginally superior to an increasing or constant scheme, with more pronounced benefit for faster growing tumors, (2) the minimum dose to stop tumor growth is age dependent, and (3) a dose-dense schedule is favored. Faster growing tumors respond better to dose density.     

Proteomic analysis of doxorubicin‐induced changes in the proteome of HepG2cells combining 2‐D DIGE and LC‐MS/MS approaches

HepG‐2 cells are widely used as a cell model to investigate hepatocellular carcinomas and the effect of anticancer drugs such as doxorubicin, an effective antineoplastic agent, which has broad antitumoral activity against many solid and hematological malignancies. To investigate the effect of doxorubicin on the protein pattern, we used complementary proteomic workflows including 2‐D gel‐based and gel‐free methods. The analysis of crude HepG2 cell extracts by 2‐D DIGE provided data on 1835 protein spots which was then complemented by MS‐centered analysis of stable isotope labeling by amino acids in cell culture‐labeled cells. The monitoring of more than 1300 distinct proteins, including proteins of the membrane fraction provides the most comprehensive overview on the proteome of the widely used model cell line HepG2. Of the proteins monitored in total, 155 displayed doxorubicin‐induced changes in abundance. Functional analysis revealed major influences of doxorubicin on proteins involved in protein synthesis, DNA damage control, electron transport/mitochondrial function, and tumor growth. The strongest decrease in level was found for proteins involved in DNA replication and protein synthesis, whereas proteins with a function in DNA damage control and oxidative stress management displayed increased levels following treatment with doxorubicin compared with control cells. Furthermore, the doxorubicin‐associated increase in levels of multiple forms of keratins 8, 18, and 19 and other structural proteins revealed an influence on the cytoskeleton network.     

Silencing of Bmi-1 gene by RNA interference enhances sensitivity to doxorubicin in breast cancer cells

The oncogene Bmi-1 is highly up-regulated in breast carcinoma and is found to be efficient in preventing apoptosis of the cancer cells. Doxorubicin is an important chemotherapeutic agent against breast carcinoma. However, the effective therapeutic response to doxorubicin is often associated with severe toxicity. The present study is targetted at developing a strategy to increase doxorubicin sensitivity to lower doses without compromising its efficacy. A stable cell line with a persistent silencing of Bmi-1 was established. MTT assay was performed to evaluate 50% inhibitory concentration (IC50) values of doxorubicin. Apoptosis was detected by FCM and the expression of related genes [phosphor-Akt (pAkt), totle-Akt (tAkt), Bcl-2 and Bax] was studied by Western blot. In vivo, the sensitivity of the tumor tissues against doxorubicin was evaluated by transplanted MCF-7 nude mice model and the apoptosis of tissue cells was detected by TUNEL assay. The expression of pAkt and Bcl-2 was down-regulated, whereas Bax was up-regulated in Bmi-1 silencing cells. The results obtained indicated that silencing of Bmi-1 can render MCF-7 cells more sensitive to doxorubicin which induced a significantly higher percentage of apoptosis cells in vitro and in vivo. All together these results clearly demonstrate that Bmi-1 siliencing combined treatment of doxorubicin might be a new strategy for biological treatment on breast cancer.     

A peptide-doxorubicin 'prodrug' activated by prostate-specific antigen selectively kills prostate tumor cells positive for prostate-specific antigen in vivo

We covalently linked doxorubicin with a peptide that is hydrolyzable by prostate-specific antigen. In the presence of prostate tumor cells secreting prostate-specific antigen, the peptide moiety of this conjugate, L-377,202, was hydrolyzed, resulting in the release of leucine-doxorubicin and doxorubicin, which are both very cytotoxic to cancer cells. However, L-377,202 was much less cytotoxic than conventional doxorubicin to cells in culture that do not secrete prostate-specific antigen. L-377,202 was approximately 15 times more effective than was conventional doxorubicin at inhibiting the growth of human prostate cancer tumors in nude mice when both drugs were used at their maximally tolerated doses. Nude mice inoculated with human prostate tumor cells secreting prostate-specific antigen showed considerable reductions in tumor burden with minimal total body weight loss when treated with L-377, 202. This improvement in therapeutic index correlated with the selective localization of leucine-doxorubicin and free doxorubicin in tissues secreting prostate-specific antigen after exposure to L-377,202.     

Optimal and suboptimal protocols for a class of mathematical models of tumor anti-angiogenesis

Tumor anti-angiogenesis is a cancer treatment approach that aims at preventing the primary tumor from developing its own vascular network needed for further growth. In this paper the problem of how to schedule an a priori given amount of angiogenic inhibitors in order to minimize the tumor volume is considered for three related mathematical formulations of a biologically validated model developed by Hahnfeldt et al. [1999. Tumor development under angiogenic signalling: a dynamical theory of tumor growth, treatment response, and postvascular dormancy. Cancer Res. 59, 4770-4775]. Easily implementable piecewise constant protocols are compared with the mathematically optimal solutions. It is shown that a constant dosage protocol with rate given by the averaged optimal control is an excellent suboptimal protocol for the original model that achieves tumor values that lie within 1% of the theoretically optimal values. It is also observed that the averaged optimal dose is decreasing as a function of the initial tumor volume.     

Therapeutic metformin/AMPK activation blocked lymphoma cell growth via inhibition of mTOR pathway and induction of autophagy

Adenosine monophosphate-activated protein kinase (AMPK) acts as a major sensor of cellular energy status in cancers and is critically involved in cell sensitivity to anticancer agents. Here, we showed that AMPK was inactivated in lymphoma and related to the upregulation of the mammalian target of rapamycin (mTOR) pathway. AMPK activator metformin potentially inhibited the growth of B- and T-lymphoma cells. Strong antitumor effect was also observed on primary lymphoma cells while sparing normal hematopoiesis ex vivo. Metformin-induced AMPK activation was associated with the inhibition of the mTOR signaling without involving AKT. Moreover, lymphoma cell response to the chemotherapeutic agent doxorubicin and mTOR inhibitor temsirolimus was significantly enhanced when co-treated with metformin. Pharmacologic and molecular knock-down of AMPK attenuated metformin-mediated lymphoma cell growth inhibition and drug sensitization. In vivo, metformin induced AMPK activation, mTOR inhibition and remarkably blocked tumor growth in murine lymphoma xenografts. Of note, metformin was equally effective when given orally. Combined treatment of oral metformin with doxorubicin or temsirolimus triggered lymphoma cell autophagy and functioned more efficiently than either agent alone. Taken together, these data provided first evidence for the growth-inhibitory and drug-sensitizing effect of metformin on lymphoma. Selectively targeting mTOR pathway through AMPK activation may thus represent a promising new strategy to improve treatment of lymphoma patients.