23.12.2015

Bmc cancer journal impact factor 2013

AbstractBackgroundOvarian carcinoma is a rarely curable disease, for which new treatment options are required.
AbstractBackgroundFor the further development of palliative care, it is relevant to gain insight into trends in non-acute mortality. As agents that block HMG-CoA reductase and the mevalonate pathway, the statin family of drugs are used in the treatment of hypercholesterolemia and have been shown to trigger apoptosis in a tumor-specific manner. Recent clinical trials show that the addition of statins to traditional chemotherapeutic strategies can increase efficacy of targeting statin-sensitive tumors. Our goal was to assess statin-induced apoptosis of ovarian cancer cells, either alone or in combination with chemotherapeutics, and then determine these mechanisms of action.MethodsThe effect of lovastatin on ovarian cancer cell lines was evaluated alone and in combination with cisplatin and doxorubicin using several assays (MTT, TUNEL, fixed PI, PARP cleavage) and synergy determined by evaluating the combination index. Stroke accounted for 10,000 deaths, dementia for 8,000 deaths and COPD and heart failure each accounted for 6,000 deaths.
The mechanisms of action were evaluated using functional, molecular, and pharmacologic approaches.ResultsWe demonstrate that lovastatin induces apoptosis of ovarian cancer cells in a p53-independent manner and synergizes with doxorubicin, a chemotherapeutic agent used to treat recurrent cases of ovarian cancer.
Compared to 1996, the number of people who died from chronic diseases has risen by 6%.Of all non-acute deaths, almost three quarters were at least 70 years old when they died.
Lovastatin drives ovarian tumor cell death by two mechanisms: first, by blocking HMG-CoA reductase activity, and second, by sensitizing multi-drug resistant cells to doxorubicin by a novel mevalonate-independent mechanism.
Almost one third of the people died at home (31%), 28% in a hospital, 25% in a nursing home and 16% somewhere else.ConclusionFurther investments to facilitate dying at home are desirable. This inhibition of drug transport, likely through inhibition of P-glycoprotein, potentiates both DNA damage and tumor cell apoptosis.ConclusionsThe results of this research provide pre-clinical data to warrant further evaluation of statins as potential anti-cancer agents to treat ovarian carcinoma.
Death certificate data proved to be useful to describe and monitor trends in non-acute deaths. Many statins are inexpensive, off-patent generic drugs that are immediately available for use as anti-cancer agents. We provide evidence that lovastatin triggers apoptosis of ovarian cancer cells as a single agent by a mevalonate-dependent mechanism. Moreover, we also show lovastatin synergizes with doxorubicin, an agent administered for recurrent disease. This synergy occurs by a novel mevalonate-independent mechanism that antagonizes drug resistance, likely by inhibiting P-glycoprotein. For the planning and organisation of palliative care it is important to gain insight into the background characteristics of people who die from cancer or other chronic diseases. While patients typically undergo a period of remission of 1-2 years, more than half eventually relapse. This is especially important in the context of an ageing population, which will ultimately lead to a greater number of deaths.In the year 2000 the authors performed a first analysis of non-acute death.
This update was necessary because the previous study concerned mortality data that are now ten years old. Statins inhibit the rate-limiting enzyme of the mevalonate (MVA) pathway, HMG-CoA reductase (HMGCR), and have been used for decades as safe and effective agents in the control of hypercholesterolemia[7, 8] In addition to cholesterol, the MVA pathway gives rise to a number of crucial biochemical end-products, including ubiquinone, dolichol, isopentenyladenine, and isoprenoid precursors. It was also important because on a national and international level palliative care is seen as a spearhead of healthcare policy. Statins can trigger tumor cells to undergo a classic caspase-dependent, apoptotic response that is reversible by exogenous addition of MVA or the isoprenoid precursors, geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP)[7]. Thus, the statin family of drugs are immediately available for use as part of the arsenal of molecular targeted therapeutics to combat cancer.Like most anti-cancer agents, statins demonstrate robust efficacy on some but not all tumor-types, emphasizing the importance of matching the agent with the sensitive, responsive cancer. Statins have been extensively shown to trigger apoptosis of cell lines derived from haematological malignancies, including acute myelogenous leukemia and multiple myeloma[7, 9]. For the period 2003a€“2006, also the place of death could be obtained.The Netherlands, as other western countries, has an obligatory death reporting system in which the physician who treated the deceased at the time of death or a pathologist fills out the death certificate. Very recently, preliminary reports have indicated that ovarian carcinoma is sensitive to statin-induced apoptosis, providing a unique alternative to treating this deadly disease[14, 15].To advance these findings, we demonstrate that lovastatin induces apoptosis of ovarian cancer cells in a p53-independent manner and synergizes with doxorubicin, a chemotherapeutic agent used to treat recurrent ovarian cancer.
When filling out this certificate the physician has to follow the cause-of-death certification guidelines of the World Health Organisation (WHO). Physicians are asked to give up to 4 causes of death, with one of them being the "primary" or "underlying" cause that started the chain of events leading to death. Effects of complications of the disease that is considered as the "primary" cause are called "secondary" causes.All causes mentioned by the physician are coded by coders at the CBS in accordance with the WHO coding guidelines. The 10-th edition of the International Classification of Diseases (ICD-10) is used to code the causes of death from 1996 onwards.Statistics on deceased people are regularly published by Statistics Netherlands, with in general a one year time lag. Treatments of 20 I?M lovastatin or vehicle control were completed for 24 or 48 hours before cells were harvested for PARP (Cell Signaling Technology), Rap1 (Santa Cruz Biotechnology), p53 (Santa Cruz Biotechnology), actin (Sigma-Aldrich), and tubulin (Calbiochem) immunoblotting as described[9, 17] Lysates from cells exposed to 8 GY of radiation for 8 hours were immunoblotted for p21 (Santa Cruz Biotechnology) and tubulin.
In addition, Statistics Netherlands offers researchers the opportunity to do scientific research a€“ under strict conditions, related to protecting the anonymity of the deceased individuals a€“ by using the data in their databases.We gathered several aggregated tables with number of deaths broken distinguished by year of death (1996a€“2006), gender (Male-Female), age at death (5- or 10-year age groups), cause of death (2- or 3-digit ICD-10 codes) and, for 2003a€“2006, place of death (hospital, nursing home, home for the elderly, home or other).
Most of these tables were downloaded from Statline, but the tables with place of death were delivered on demand by Statistics Netherlands.The aggregated tables contained no data that could be reduced to individuals. Therefore, according to Dutch law ethical approval for this study was not needed.Within the total set of death certificate data we selected data from people with a chronic disease as primary cause of death (table 1). CEMVBL cells were similarly plated and treated with lovastatin and doxorubicin, also centered on each drug's MTT50 (42 and 68 I?M, respectively).
The selected diseases represent the main chronic diseases, with at least 50 people who have died from such a specific chronic condition in the Netherlands.
Cells were washed with staining buffer, resuspended in 1 mL buffer, and fluorescence in the FL1 channel was detected by flow cytometry. In a little over 40,000 deceased people cancer was the primary cause of death (see figure 1).
10 000 events were captured for analysis with Cell Quest software.Measurement of intracellular doxorubicinIntracellular doxorubicin was measured as described previously[23]. The number of people who died from any of the other selected chronic diseases was considerably lower. Stroke accounted for 10,000 deaths, dementia for 8,000 deaths and COPD and heart failure each for 6,000 deaths.Figure 1Number of deaths in the Netherlands from chronic diseases in 2006 according to primary cause of death.
Briefly, for accumulation experiments, 2 A— 105 A2780ADR cells were seeded in 6-well plates for 24 hours and treated.
The spread among the age groups does vary somewhat depending on the chronic disease (not shown here). An additional sample was treated with doxorubicin, 10 I?M lovastatin, and 100 I?M MVA as well. For example, in the case of people who died from cancer a peak is seen in the 75a€“80 years of age group. For stroke, COPD, diabetes, Parkinson's disease and kidney diseases a peak is seen in the 80a€“85 years of age group and for dementia and heart failure in the 85a€“90 years of age group.Figure 2Number of deaths in the Netherlands from chronic diseases in 2006 according to age.
An additional sample was treated with doxorubicin, 21 I?M lovastatin, and 100 I?M MVA as well. The proportion of women appears to vary with age: in the 30a€“50 years of age group the proportion of women within the people who die from a chronic disease is consistently around 55%. For both accumulation and retention experiments, after treatment, cells were washed twice in cold phosphate buffered saline (PBS), resuspended in 1 mL of cold PBS, and filtered for single cells on ice.
From the age of 50 the proportion of women decreases to about 40% for the 65a€“70 and 70a€“75 years of age groups and then increases to 81% for the highest age group.Figure 3Percentage females among deaths from chronic diseases in the Netherlands in 2006 according to age.
Comet assays were performed as described previously[24] Briefly, comets were visualized by fluorescence microscopy (Axioskop microscope, Zeiss) and images were analyzed using Komet 5.5 software (Kinetic Imaging). Briefly, 2 million cells were seeded in 100 mm plates for 24 hours and treated with the indicated drugs for 24 hours to determine both the proportion of TUNEL-positive cells and the cell cycle phase from which apoptotic cells arose.ResultsHuman ovarian carcinoma cells are sensitive to lovastatin-induced apoptosisTo evaluate the sensitivity of human ovarian cancer cells to the anti-proliferative activity of statins, a panel of ten ovarian cancer cell lines was exposed to increasing concentrations of lovastatin for 48 hours. See figure 4 for the developments in mortality related to other chronic diseases.Figure 4Percentage change in deaths from chronic diseases in the Netherlands from 1996a€“2006 according to primary cause of death. A series of experiments were conducted to define the cytotoxic effects of lovastatin on a panel of human ovarian cancer cell lines. A: Cells were exposed to a range of lovastatin concentrations for 48 hours to determine MTT50 values by MTT assay.
MTT50s are presented as the mean of 2-4 independent experiments with error bars representing standard deviation. B: Cells were exposed to 20 I?M lovastatin or vehicle control for 24 or 48 hours to assess the presence of PARP cleavage by western blotting. For each of the different chronic diseases, different trends or changes in trends are visible. For cancer, the number of deaths slightly increased almost each year with about 1%, but showed a small decrease in 1997 and 2000.
For fixed PI, cells were seeded and treated as above to 20 I?M lovastatin or vehicle control. Results are normalized to the ethanol controls (= 1%) and data are presented as the mean of 2-4 independent experiments with error bars representing standard deviation.
C: Cells were seeded as above, exposed to 20 I?M lovastatin or a vehicle control for 24 hours, lysed and immunoblotted to detect processed (P) and unprocessed (U) Rap1 and actin.
Experiments were conducted 2-3 times and representative blots are shown.To further define the antiproliferative effect of lovastatin, we next used two independent methods to evaluate whether lovastatin triggered apoptosis in the cell line panel. We first assessed the population of pre-G1 cells by fixed PI flow cytometry in response to 20 I?M lovastatin for 48 hours. It may be assumed that 'other' includes, for instance, non-acute deaths in institutions for the mentally handicapped, mental healthcare institutions or one the 230 independent hospices that exits nowadays in the Netherlands. Ten years ago, the number of independent hospices was only some 40.Figure 5Place of death for people who died from a chronic disease in the Netherlands in 2006 according to gender. In all ten cell lines tested, an increase in the pre-G1 population was measured (Figure 1B, histograms, and data not shown).
As an independent assay for apoptosis we assessed whether the cleavage of poly ADP-ribose polymerase (PARP) was detectable in cells treated with either lovastatin or vehicle control for 48 hours. The proportion of men who died from a chronic disease in a nursing home or home for the elderly, however, is much smaller than the proportion of women: 20% and 7% compared to 30% and 14%, respectively.


Cleaved PARP was observed in all cell lines, except DOV13 (Figure 1B, inset western blots, and data not shown). Thus, ovarian cancer cells undergo apoptosis in response to lovastatin exposure.Treating cells with 20 I?M lovastatin for 48 hours elicited a robust apoptotic response from which a potent MVA-dependent rescue would be required. For the non-acute deaths of people 70 years of age and older the proportion who died at home gradually decreased with age. At comparable ages fewer women than men died at home.Developments in place of deathAs Statistics Netherlands did not collect data on place of death until 2003, developments in the place of death in case of non-acute deaths are only analysed for the period 2003a€“2006 (see figure 6). MVA reversed the effect of lovastatin and both GGPP and FPP were also able to partially rescue cells (Figure 1B, and data not shown). The proportion of people who died in a hospital from one of the specified chronic diseases appears to have decreased somewhat in the past four years, from 31% in 2003 to 28% in 2006. The mechanism of lovastatin-induced apoptosis in ovarian cancer cells is therefore dependent upon HMGCR inhibition.To ensure that the MVA pathway block was due to lovastatin we evaluated the prenylation status of Rap1, a protein that is known to be exclusively geranylgeranylated (Figure 1C). The proportion of people who died in a nursing home has, at the same time, increased somewhat, from 23% in 2003 to 25% in 2006.Figure 6Place of death for people who died from a chronic disease in the Netherlands in 2003a€“2006.
Immunoblot analysis showed accumulation of the unprocessed form of Rap1 in all cell lines exposed to lovastatin for 24 hours, indicating that drug uptake was universally achieved.Lovastatin triggers apoptosis of human ovarian carcinoma cells in a time and dose dependent mannerNovel treatment options for women diagnosed with ovarian cancer are sorely needed. Our data suggests that statins have potential to be used as chemotherapeutics for these patients but it is important to determine whether they are effective at therapeutically achievable (low micromolar) concentrations. As a representative sensitive ovarian cancer cell line, A2780 cells were exposed to increasing concentrations of lovastatin (1, 5, 10, and 20 I?M) for 24, 48, and 72 hours before being harvested for fixed PI analysis and the population of pre-G1 cells measured (Figure 2). The number of 77,000 may be a small underestimation of non-acute deaths as smaller diagnosis groups have not been included in our analyses. But it could also be an overestimate, because some people who had a chronic disease as primary cause of death, could in fact have died of acute complications of these chronic diseases.In a study by Van der Heide et al.
A2780 cells were exposed to a range of lovastatin concentrations or vehicle control for 24, 48, or 72 hours to demonstrate the dose- and time- dependence of lovastatin-induced apoptosis as measured by fixed PI analysis. Results are normalized to each vehicle control (= 1%) and are presented as the mean of 3 independent experiments with error bars representing standard deviation. Dashed line highlights equivalence between doses of 20 I?M for 24 hours and 5 I?M for 72 hours. Taking a molecular approach, a dominant negative p53 truncation, p53DD, was ectopically expressed in A2780 cells (Figure 3A).
Particularly in very old people whose condition gradually deteriorates death is almost never entirely unexpected and acute, even though most patients will probably die from another cause than one of our specified chronic diseases. This indicates that the way in which non-acute deaths have been operationalised and assessed needs to be taken into account when interpreting the sometimes divergent non-acute death percentages.Selection of diseasesThe way in which we operationalised the term 'non-acute deaths', is open for discussion. The activity of p53 in these cells was evaluated by exposing them to 8 GY of gamma-irradiation measuring p53-dependent induction of the cyclin-dependent kinase inhibitor, p21. Hereby we also included people who died from an acute complication of a chronic disease as an underlying cause.
A: A2780 YFP and A2780 p53DD cells were harvested, lysed, and immunoblotted with anti-p53 antibody to detect p53DD and with anti-tubulin as a loading control.
We intend to repeat our study using multiple causes, which are divided between primary and secondary causes. B: A2780 YFP and p53DD cell were seeded and then exposed to 8 GY of radiation for 8 hours, harvested, lysed, and immunoblotted for the detection of p21 and tubulin. Then we can make a distinction between people dying from acute or chronic complications of chronic diseases.We also had a somewhat arbitrary exclusion of people who died from some chronic diseases.
C: A2780 pBP and Bcl-2 cells were seeded as above, harvested, lysed, and immunoblotted with antibodies to Bcl-2 and tubulin. Although most of these excluded chronic diseases are quite small, some categories account for a substantial amount of up to several hundreds or more. For chronic kidney diseases one could think of including N05 (unspecified nephritic syndrome) or N19 (unspecified renal failure).
These would add another 10 respectively 327 deaths to the 903 deaths from chronic kidney diseases mentioned in this article for 2006. We also could have included all "sequelae" (or "late consequence") codes as a non-acute cause of death, like B90 (Sequelae of tuberculosis, with 45 deaths), G09 (Sequelae of inflammatory diseases of the central nervous system, with 5 deaths) or I69 (Sequelae of cerebrovascular disease, with 522 deaths).In the intended repetition of this study, we aim to look at heart diseases more thoroughly. Data are presented as the mean of 6 independent experiments with error bars representing standard deviation.
Chronic heart diseases, like I25 (chronic ischemic heart diseases) result both in acute and chronic complications amenable to palliative care.In a Dutch publication on this study [19] we decided to include for stroke only 67% of the deaths to account for 33% acute deaths. This was a very crude estimate and it could be argued that we overestimated the acute deaths for stroke in that publication. Fixed PI was used to measure the pre-G1 population of cells treated with 20 I?M lovastatin for 24 hours (Figure 3D). Empty vector controls and cells expressing p53DD underwent a similar degree of apoptosis while ectopic expression of Bcl-2 significantly decreased the amount of lovastatin-induced apoptosis by 50%, demonstrating the p53-independence of lovastatin-induced apoptosis in human ovarian cancer cells.Lovastatin synergizes with doxorubicin in P-gp expressing cellsAs statins will likely be used in cancer treatment as part of drug cocktails, we next evaluated whether the addition of lovastatin to agents presently used in chemotherapy regimes of ovarian cancer would increase efficacy. Cisplatin and doxorubicin were selected as representative agents used in the treatment of primary and relapsed drug-resistant ovarian cancer. To model primary onset disease A2780 cells were treated in combination with cisplatin and lovastatin.
To model relapsed disease A2780ADR cells, a multi-drug resistant cell line derived from parental A2780 cells, were treated with lovastatin in combination with either cisplatin or doxorubicin.
Synergy experiments were adapted from the methods described by Chou and Talaly [21] to determine the antiproliferative effect of drug combinations with lovastatin. In A2780ADR cells, lovastatin and cisplatin were additive (C = 1) when combined at higher concentrations (Figure 4A, white bars). And we did not pay any attention to the amount or content of the palliative care that people used or are looking for.Place of deathInsight into the place of death is important, among other things, because it is increasingly regarded as an important quality indicator for good palliative care.
A2780ADR (A) and CEMVBL (B) cells were plated for MTT assays and treated for 24 hours with concentration ranges of lovastatin and either doxorubicin or cisplatin. Combination index (CI) plots were generated using CalcuSyn software to determine whether the drugs synergize (CI < 1).
Data is presented as the mean of 5-10 independently determined CI values with error bars representing standard deviation. C: Substantially higher P-gp expression was detected by flow cytometry in A2780ADR and CEMVBL cells compared to A2780 and CEM cells, respectively, using a fluorescence-tagged anti-P-gp antibody. Our time frame was too small to show trends.When someone dies in hospital it is very likely that some healthcare transitions have taken place in the final stages. We hypothesized that P-gp mediated efflux of doxorubicin, a known substrate of P-gp, was being blocked by lovastatin via an unknown mechanism.
Such transitions may bring about unrest and discomfort for both the patient and his family. To verify that synergy between lovastatin and doxorubicin was not simply an artifact of the A2780ADR cell system, we employed an alternative paired parental and MDR model derived from acute lymphoblastic leukemia, CEM and CEMVBL cells, respectively.
We also confirmed that the CEMVBL cells both overexpress P-gp on their cell surface and have a significantly higher MTT50 for doxorubicin when compared to the parental CEM cells (Figure 4C, right, and data not shown). Other advantages are that the data are population based, relatively easy to obtain and at relatively little cost.The usability of death certificate data to analyse trends in mortality on a national level has not only been established in our study, but for instance also in a study regarding ten-year trends in the place of death of cancer patients in England by Higginson et al. Interestingly, lovastatin synergized significantly with doxorubicin in CEMVBL cells (Figure 4B) using the same experimental design as above. It is impossible, for instance, to be absolutely certain whether the cause of death as stated on the death certificate is in fact the real cause of death, because questions can remain considering the underlying causes, even after an autopsy. As the fluorescence of doxorubicin can be directly measured by flow cytometry, we evaluated the amount of doxorubicin within A2780ADR and CEMVBL cells exposed to a sub-lethal dose of doxorubicin alone or in combination with increasing concentrations of lovastatin. We are also not absolutely certain whether the death certificate data completely correspond with the data in medical records.
Previous research often focused on either a very specific population, such as persons who died from cancer, [4, 12, 13] or on the total deceased population. A2780ADR (A) and CEMVBL (B) cells were treated with lovastatin and doxorubicin as indicated for 3 hours, with or without MVA.
Intracellular doxorubicin fluorescence was measured by flow cytometry and the results are normalized to cells treated with doxorubicin alone (= 100%). As the ICD-10 is always used when death certificates are coded, it is relatively easy to make selections and thus to focus on the groups of patients that are most relevant within the scope of the planning and organisation of palliative care.
Data are presented as the mean of 3-4 independent experiments with error bars representing standard deviation. It is interesting to find out in future research in other countries but in the same selection of causes of death, whether different or similar trends can be observed. Combining cross-years and cross-national comparisons of place of death and other socio-demographic data of people who have died from chronic diseases can be helpful to plan and monitor national and international policies with regard to palliative care.ConclusionFurther investments to facilitate dying at home are desirable.
Advantages of the use of death certificate data concern the reliability of the data, the opportunities for selection on the basis of the ICD-10, and the availability and low cost price of the data.AcknowledgementsThe study was financed by the Dutch ministry of public health, welfare and sports.
Intracellular doxorubicin was determined by flow cytometry and the results are normalized to cells co-treated with doxorubicin and lovastatin without further incubation (= 100%).
Data are presented as the mean of 3 independent experiments with error bars representing standard deviation.
In this experiment, cells were treated with lovastatin and doxorubicin together to "load" the cells with doxorubicin.
To determine differential degrees of doxorubicin retention, cells were further incubated for 2 hours in doxorubicin-free media with or without lovastatin. DW was responsible for the selection of "chronic diseases" and helped draft the manuscript. Remarkably, incubation with lovastatin resulted in more intracellular doxorubicin remaining after 2 hours. Partial loss of doxorubicin observed in cells that were incubated with lovastatin is likely due to passive diffusion or efflux mediated by alternative mechanisms because this same pattern was observed in parental A2780 cells, which do not overexpress P-gp, treated in the same manner (data not shown).


These data suggest that lovastatin may inhibit P-gp from actively pumping doxorubicin out of the cell. Surprisingly, lovastatin-induced accumulation of doxorubicin was not reversed by co-incubation with MVA (Figure 5A; B), suggesting that a mechanism independent of HMGCR inhibition is at work. Drug concentrations used in this set of experiments were relatively sub-lethal, half-MTT50 values to minimize the effect of each drug on its own. Although these doses are higher than physiologically achievable levels, they remain experimentally tractable.
While doxorubicin exposure alone resulted in a slight, significant increase in DNA damage compared to either control- or lovastatin-treated cells, combined treatment with both lovastatin and doxorubicin together resulted in a statistically significant 3-fold increase in DNA damage over doxorubicin alone (Figure 6A).Figure 6Lovastatin potentiates DNA damage and apoptosis induced by doxorubicin. A: Comet assays were performed and the olive tail moment determined for 75 cells of each condition. Data are presented as the mean of 4 independent experiments with error bars representing standard deviation. TUNEL and PI dual-staining was carried out to determine the proportion of TUNEL-positive cells and if apoptotic cells originated predominantly in any particular phase of the cell cycle. B: Data are presented as the mean of 3 independent experiments measuring either TUNEL positivity (left) or PreG1 population (right) with error bars representing standard deviation.
For these experiments we used dual-staining of TUNEL and fixed PI to measure the degree of apoptosis and determine if cells undergo apoptosis preferentially from any particular phase of the cell cycle. Similar to the comet assays, doxorubicin alone induced a small increase in apoptosis compared to either the control- or lovastatin-treated cells (Figure 6B). Cells treated with lovastatin alone, however, showed no evidence of either DNA damage or apoptosis. Conversely, cells exposed to the combination of lovastatin and doxorubicin underwent a statistically significant 10-fold increase in apoptosis when compared to doxorubicin alone. While over-expression of Bcl-2 did not inhibit the combined activity of lovastatin and doxorubicin (Additional file 2: Supplemental Figure S2), apoptotic cells were detected from all phases of the cell cycle (Figure 6C). Therefore, doxorubicin and lovastatin combine synergistically to induce high levels of both DNA damage and apoptosis in human ovarian cancer cells.DiscussionOur work provides important evidence to support further pre-clinical and clinical evaluation of the statin family of drugs as anticancer agents against ovarian cancer. We show that a panel of ovarian cancer derived cell lines is sensitive to lovastatin-induced apoptosis, consistent with recent reports[14, 15]. Mechanistically this apoptotic pathway is functionally blocked by exogenous MVA or the isoprenoid precursors GGPP and FPP.
Moreover, we show that statin killing occurs irrespective of the mutational status of the tumor suppressor p53.
Our results using a dominant negative p53 clearly indicate that lovastatin-induced apoptosis is substantially p53-independent and this is also supported by the observation that p53-null SKOV3 cells are able to undergo lovastatin-induced apoptosis. These observations are particularly important for ovarian cancer in which p53 mutation rates have been estimated between 23 and 79%[32]. Synergy is achieved by lovastatin blocking drug efflux through a MVA-independent mechanism that enables the intracellular retention and genotoxic action of doxorubicin.
To the best of our knowledge, these latter features of statin-induced apoptosis have not yet been reported for ovarian cancer. Exploiting the unique ability of statins to drive apoptosis by the mevalonate-dependent mechanism alone warrants further evaluation of these agents in the treatment of ovarian cancer (Figure 6D, left side). In addition, using statins, like lovastatin, to synergize with chemotherapeutics that are P-gp substrates (Figure 6D, right side) may be a feature of lovastatin action that further maximizes ovarian cancer cell death and improves patient survival.It is interesting to note that while several reports have shown that P-gp expressing cells were more sensitive statin-induced apoptosis, [33a€“36] our results show the opposite trend (Figure 1A).
Indeed, the MTT50 results for lovastatin in A2780ADR and A2780CIS cells are roughly 5-fold higher than in the parental A2780 cells.
The reason for this difference is unknown, but it is possible that the drug resistant cells have exploited additional mechanisms of resistance, such as increasing the expression of anti-apoptotic proteins.As agents approved for use in humans, the MVA-dependent antiproliferative activity of statins has prompted several Phase I clinical trials of statins on a wide variety of late-stage cancers, and although statins were well tolerated, only limited responses were evident. More recently statins have been evaluated on cohorts of patients harboring a tumor-type that has been shown to be sensitive to statin-induced apoptosis in tissue culture studies. In these focused, tumor-specific, hypothesis-driven trials, statins have demonstrated some efficacy as a single agent[29, 30, 37] but more wide-reaching effects were evident when statins were combined with chemotherapeutics [10, 11, 38, 39]. Thus, our data identifying ovarian carcinoma as a statin-sensitive tumor type strongly supports the evaluation of statins in strategies to combat this disease.A recent, retrospective epidemiological study showed that statin use in patients diagnosed with epithelial ovarian cancer is associated with improved survival[40].
Although only a relatively small number of patients met the criteria for the study, multivariable analysis identified statin use as an independent positive prognostic factor after controlling for age, stage, grade, and suboptimal cytoreduction, providing clinical support for the use statin-based combinations in cancer treatment. For example, it appears that lipophilic statin use after breast cancer diagnosis has been associated with decreased risk of recurrence[25, 26]. Overall, these recent studies provide supporting rationale for the use of statins as anticancer agents and suggest that lipophilic statins (lovastatin, simvastatin, atorvastatin, fluvastatin, and pitavastatin) may be more effective, perhaps because they are better able to penetrate solid tumors compared to hydrophilic statins. From a pharmacological perspective, the lipophilic statins that demonstrate higher plasma concentrations with longer retention times in the circulation include atorvastatin and fluvastatin. This suggests these lipophilic agents may best target the tumor and show higher anti-cancer efficacy in vivo, consistent with a previous study comparing lipophilic and hydrophilic statins in ovarian cancer[15].Recent evidence suggests that there may be a connection between drug resistance and regulation of the MVA pathway. In MDR AML cells, HMGCR mRNA levels were not significantly elevated upon statin exposure in cells that showed preferential sensitivity to lovastatin[36]. More recently, it was suggested that high levels of HMGCR mRNA correlates with sensitivity to statin-induced apoptosis[15].
It will be interesting in the future to determine how HMGCR expression impacts statin sensitivity and whether it can be exploited as a biomarker.Mechanistically, it is clear that statins target HMG-CoA reductase and similarly trigger tumor cells to undergo apoptosis[7]. Several classes of specific P-gp inhibitors have been developed but have unfortunately shown general cytotoxicity in clinical trials[42]. This is thought to be due to targeting P-gp not only on tumor cells, but also on several normal vital organs that constitutively express P-gp. It would be easy to assume that statins blocking P-gp will similarly cause general cytotoxicity, however, it is not known whether statins and classic P-gp inhibitors are mechanistically or functionally similar. Lovastatin has been reported to inhibit P-gp in a limited number of biochemical studies with two very distinct caveats: none have used human cells overexpressing drug-selected human P-gp and the concentrations of drug used have been well beyond the physiologically achievable range [43a€“46]. Moreover, the results of these studies have been in conflict when using either the acid or lactone form of the statin[45, 46]. While the reasons for this result are unclear, it is possible that the cells have become drug-resistant through means other than the MDR machinery, such as upregulation of one or more anti-apoptotic proteins, and thereby rendered forced expression of Bcl-2 incapable of rescuing cells further.
Further study will be required to better understand the interplay of all mechanisms of drug resistance.Statins ultimately need to advance to clinical trials where their inhibition of drug efflux can be monitored on both tumor and normal cells. Interestingly, other groups have reported that lovastatin protects normal cells from doxorubicin-induced cytotoxicity [47a€“49] which, when combined with our data, suggests that statins may affect P-gp differently in normal cells compared to tumor cells.
It is entirely possible that lovastatin functionally blocks P-gp in a manner that is distinct from classic P-gp inhibition. Evidence that statins can be successfully combined with various P-gp substrates is also established from their safe and effective combination in the polypharmacy of cardiac patients with hypercholesterolemia[50]. Taken together, our results suggest the ability of statins to trigger apoptosis of ovarian cancer cells may be exploited in the treatment of this disease, and that the potential P-gp inhibitory properties of certain statins, like lovastatin, warrant further investigation. It is also of interest to note that at MTT50 concentrations, but not higher, lovastatin had a slightly antagonistic relationship with cisplatin, a non-P-gp substrate (Figure 4A, Additional file 1: Supplemental Figure S1). This observation suggests that it could potentially be deleterious to combine lovastatin with cisplatin in the treatment of some patients. Furthermore, lovastatin and doxorubicin were also able to synergize in A2780 parental and A2780CIS cells. While this suggests that elements other than P-gp are involved in the interaction between these two drugs, the degree of synergy observed in A2780ADR cells is higher, indicating that inhibition of P-gp is likely an important mechanism of how lovastatin synergizes with doxorubicin. These results require further investigation to truly understand the manner by which lovastatin functionally interacts with other chemotherapeutics.Determining which statin will maximally target different tumors, including ovarian, under different conditions will also be vital to advancing patient care. In the 14 completed and 20 or more ongoing clinical trials evaluating statins in the prevention or treatment of cancer, [10a€“12, 30, 37a€“39, 51a€“57] the rationale for choosing a particular statin is not presented and appears random.
Indeed, the ideal choice of statin as an anti-cancer agent remains unclear, however, evidence suggests lipophilic agents with pharmacologic properties that favor access to solid tumors is of high priority. Further work is required to better understand the activity of these statins as potential inhibitors of P-gp and to determine if this inhibition is specific to tumor cells in vivo.ConclusionsOverall, our results identify ovarian cancer cells as sensitive to statin-induced apoptosis and strongly suggest that statins can play a role in the treatment of ovarian carcinoma.
As approved agents, statins can make immediate impact either as additions to traditional inductive therapy, as maintenance therapy to secure lasting remissions, or as salvage treatment for terminal, refractory disease.
Our results may impact ongoing clinical trials using statins as anti-cancer agents and will be important to consider in the design of future clinical trials targeting various tumor types, including ovarian cancer.AcknowledgementsWe would like to express our profound appreciation to Dr. Sincere thanks to members of the Penn lab for helpful discussions and critical review of the manuscript as well as to the following for providing necessary reagents: Dr.
Brown (The Samuel Lunenfeld Research Institute, Toronto, ON, Canada; Skov3, Ovca429, Hey, and HOC-7 cells), Dr. Pearson (Peter MacCallum Cancer Centre, Melbourne, Australia; DOV13, Ovca432, and OVHS-1 cells), Dr. Jeremy Squire (University of Toronto, Toronto, ON, Canada; A2780, A2780ADR, and A2780CIS cells), Dr. David Andrews (McMaster University, Hamilton, ON, Canada; Bcl-2 antibody), and Apoptex Corporation (lovastatin).
Supplementary data that shows lovastatin did not synergize with cisplatin in either parental A2780 cells or drug-resistant A2780CIS cells and that lovastatin and doxorubicin were borderline synergistic or additive in A2780 and A2780CIS cells.
Supplementary data that shows Bcl-2 was unable to inhibit cell death induced by the combination of lovastatin and doxorubicin. LZP conceived of the study, participated in its design and coordination, and helped to draft the manuscript.



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