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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Cell Biochem. Author manuscript; available in PMC 2012 August 1.
Published in final edited form as:
PMCID: PMC3406746
NIHMSID: NIHMS287058

Resistance to paclitaxel increases the sensitivity to other microenvironmental stresses in prostate cancer cells

Abstract

The microenvironment is central to many aspects of cancer pathobiology and has been proposed to play a role in the development of cancer cell resistant to therapy. To examine the response to microenvironmental conditions, two paclitaxel resistant prostate cancer cell lines (stable and reversible) and one reversible heat resistant cell line were studied. In comparison to their parental cell lines, both paclitaxel resistant cell lines (stable and reversible) were more sensitive to microenvironmental heat, potentially yielding a synergistic therapeutic opportunity. In the two phenotypic cells repopulated after acute heat or paclitaxel treatments, there was an inverse correlation between paclitaxel and heat resistance: resistance to paclitaxel imparted sensitivity to heat; resistance to heat imparted sensitivity to paclitaxel. These studies indicate that as cancer cells evolve resistance to single microenvironmental stress they may be more sensitive to others, perhaps allowing us to design new approaches for prostate cancer therapy.

Keywords: prostate cancer, microenvironmental stress, drug-resistance, heat, microenvironment

Introduction

Prostate cancer (PCa) is the most common malignancy and the second leading cause of cancer-related deaths in men in the United States [Jemal et al.]. In men with advanced disease, hormonal therapy (i.e. androgen deprivation) initially induces antitumor response in more than 90% of treatment-naïve patients. However, hormonal therapy eventually fails and the PCa progresses to a castrate-resistant stage that is essentially incurable [Gopalkrishnan et al., 2001]. Chemotherapy plays an increasingly important role in the management of castrate-resistant metastatic PCa. Recently, taxanes (paclitaxel or docetaxel—DTX) in combination with other agents, such as estramustine phosphate (EMP), or dexamethasone, have been shown to result in significant antitumor responses in this population of men [Oudard et al., 2005; Petrylak et al., 2004; Tannock et al., 2004]. Although castrate-resistant PCa often initially responds to paclitaxel-based chemotherapy, the disease eventually becomes resistant. Evolution of resistance is perhaps the largest problem in cancer therapy.

In our previous studies we have established stable paclitaxel-resistant DU145-TxR and PC-3-TxR cells from DU145 and PC-3 cell lines and investigated the mechanisms of drug resistance in DU145-TxR and PC-3-TxR cells. The multiple drug resistance gene (MDR-1)-encoded P-glycoprotein was demonstrated to be overexpressed in the DU145-TxR cells, and enhanced F-actin polymerization via down regulation of CTEN expression was identified as a paclitaxel resistant mechanism in the PC-3-TxR cells [Li et al.; Takeda et al., 2007]. In addition to these molecular changes the microenvironment has been hypothesized to be a major component in the development of resistance. Microenvironmental factors, including: heat, pH, oxygen and glucose, are central factors influencing both normal and cancer cells. Many studies have documented epigenetic changes in tumor cells, and there are limited data to support that epigenetic changes in the “normal” cells may be related to the tumor microenvironment [Fiegl et al., 2006; Hu et al., 2005]. The hypoxic conditions due to high metabolic rate and abnormal blood vessels in tumors accumulate lactic acid and promote accelerated oxidation of CO2, providing an acidic extracellular tumor microenvironment (pH 5.8–7.1) [Mashima et al., 2009; Swietach et al., 2007; Vaupel et al., 1981]. In addition, glucose distribution in tumors is thought to follow similar patterns to that of oxygen. Acute and chronic hypoglycemia most likely exist in much the same way as acute and chronic hypoxia caused by transient alterations in blood supply, and diffusion-limitations, respectively [Horsman, 1995]. While oxygen and glucose concentration may be determined by diffusion into tumor tissue, pH may partly be determined by diffusion of ions out of tissue into blood vessels. Thus within the viable regions of tumors, there is a great cellular heterogeneity in oxygen concentrations, glucose content and pH values. Furthermore the tumor microenvironment may modulate apoptosis indirectly via other processes that affect apoptosis such as proliferation, repair, energy metabolism and signal transduction [Amellem and Pettersen, 1991; Belka et al., 2004; Yuan et al., 2000]. The microenvironment may also modify the radiation-induced apoptotic response of tumors [Hunter et al., 2006]. Elevation in environmental temperature has been shown to increase the cancer cell’s susceptibility to chemo- and radiation therapy [Zeng et al., 2009], and we therefore hypothesized that as the cells become resistant to one type of therapy, such as chemotherapy (taxol), they might impart sensitivity to other types of microenvironmental stresses suggesting that combination of available and new therapies might be an effective treatment approach.

In the present work, we have investigated the ability of the previously established paclitaxel resistant cell lines (PC-3-TxR and DU145-TxR) to respond to microenvironmental stresses. In addition, the responses of cell lines representing the spectrum of “normal” prostate to metastatic disease to these microenvironmental conditions were also studied. Overall these studies demonstrated that the development of chemotherapeutic resistance results in cells being less able to respond to stresses and may be the Achilles’ heel of aggressive lethal cancers.

Materials and methods

Cell lines, Culture and Isolation

The human prostate cell lines PrEC, LNCap, PC-3 and DU145 were purchased from the American Type Culture Collection (Manassas, VA). The PrEC cells were cultured in PrEBM medium with PrEGM supplements (Lonza Walkersville MD). The OPCT cells were purchased from Asterand (Detroit, MI) and cultured in phenol-red free RPMI medium with PrEGM supplements. The LN96 cell line was established previously [Pflug et al., 1999], and cultured routinely in phenol-red free RPMI supplemented with 10% charcoal-stripped (CS) FBS (Hyclone, Logan UT). The paclitaxel-resistant PC-3-TxR and DU145-TxR cells were gifts of Dr. Namiki and Dr. Mizokami (Department of Urology, Kanazawa University, Kanazawa Japan), and generated and maintained as described previously [Li et al.]. The PC-3-TxR cells were cultured in 10 nM paclitaxel (Sigma-Aldrich St. Louis, MO) to maintain their drug-resistant phenotype. Prior to each experiment, these cells were grown for a minimum of one week in normal medium. The PC-3 and PC-3-TxR cells were maintained in RPMI1640 (Sigma) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA). The DU145 and DU145-TxR cells were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA) supplemented with 10% FBS. The PC-3 and DU145 cells were treated with 100 nM paclitaxel for 48 hours and after 30 days of recovery from the treatment, the cells were harvested and designated as PCAF48 and DUAF48, respectively. PC-3 and DU145 cells were treated with 43°C heat for 24 hours and then harvested after 30 days of recovery and designated as PCAF24 and DUAF24 respectively.

Proliferation Assay

Cell growth or growth inhibition assays were performed by plating 2×105 cells in 6-well plates. After culturing for 24 hours, the cells were treated with the indicated concentrations of Paclitaxel. The cells that survived this treatment were cultured for an additional 48 hr. At the end of the culture period, the cells were trypsinized and counted using a hemocytometer. The relative cell numbers compared with untreated controls were plotted as cell viability.

Heating, Ultraviolet Radiation C (UVC), Different pH Value and Low Glucose Experiments

A typical tissue culture incubator was set to 43°C under normal humidity conditions for the heat experiments, and the UVC light (TUV 30W T8) in the cell culture hood was used for UVC exposure studies. NaOH and HCl (Sigma-Aldrich St. Louis, MO) were used to conduct the pH stress experiments, by modifying the pH values of the culture medium. The 1 mg/ml glucose concentration DMEM and no Glucose DMEM (Invitrogen, Carlsbad, CA) were utilized in the glucose stress studies.

Generation of Reversible Drug Resistant Cells

Drug-sensitive cells PC-3 and DU 145 were treated with paclitaxel, at concentrations of 100 nM for 48 hours. The viable cells remained attached on the dish at the end of 30 days recovery without paclitaxel.

Generation of Reversible Heat Resistant Cells

PC-3 and DU 145 cells were treated at 43°C for 24 hours, and the viable cells that remained attached on the dish at the end of a 30 day recovery at 37°C.

Cell Survival Assays

Cells were plated in each well of a 24-well cluster dish. Forty-eight hours after plating, media were removed and replaced with media containing 10 nM paclitaxel. Forty-eight hours later, the cells were treated at 37°C or 43°C for 2 hours, with fresh media being replaced every 2 days until untreated cells reached confluence. The media were then removed, and cells were washed with phosphate buffered saline (PBS) and then fixed with 0.5% typan blue in PBS.

Immunoblotting

Twenty-four hours after plating the cells, total protein was extracted using the M-PER Mammalian Protein Extraction Reagent (Thermo scientific, Rockford IL). Fifteen micrograms of total protein was separated using 10–20% SDS–PAGE and transferred onto PVDF membranes (Millipore, Bedford, MA), which were blocked with 5% non-fat dry milk, and immunoblotted with primary antibodies. The following primary antibodies were used: rabbit polyclonal anti-PARP, anti-caspase3, anti-NA+K+ATPase, anti-calcium binding protein CALB1, and anti-actin (Cell Signaling Technology, Inc. Danvers, MA). These primary antibodies were recognized by a goat anti-rabbit secondary antibody (Bio-Rad) and visualized using enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ).

Flow-Cytometry Assay

Expression of the CD133 surface marker was assessed in the PC-3, PCAF48, PCAF24, DU145, DUAF48 and DUAF24 cells. The cells were washed with phosphate-buffered saline (PBS) before they were removed from the culture dish with trypsin and EDTA, counted, resuspended in 5 mL of fresh medium, incubated at 37°C for 30 minutes, and then transferred to Eppendorf tubes (1×106 for PC-3 and DU145 cells, 1×104 cells per tube). After washing with PBS containing 0.5% bovine serum albumin, the cells were centrifuged at 500 G, and the supernatant was removed. The appropriate fluorochrome-conjugated antibody was added at 1:10 dilution (5–50 μL) in PBS and 0.5% bovine serum albumin and incubated on ice in the dark for 30 minutes. Briefly, 50 μL of the positive control beads and 50 μL of the negative control beads (BD Biosciences) were added, followed by 5 μL of the specific fluorochrome-conjugated antibody, and the cells were processed as described above.

Data Analysis

The statistical significance of differences in proliferation was determined by two-way ANOVA with post-hoc test. A Dunnett’s test was also performed to determine the significance of intensity differences on immunoblotting analysis; differences noted *p<0.05 were considered to be statistically significant. The data represent mean values of three replicates with standard deviation for standard errs indicated.

Results

Changing phenotypes of cells under selective pressure of microenvironmental stress factors

The key objective of this study centers on how the cell lines resistant to paclitaxel respond to the various microenvironmental stresses. To examine sensitivity of the cell lines to different stress environments, we exposed the PC-3-TxR and DU145-TxR cells along with their respective parental cells to various microenvironmental conditions. The evaluation of the resistance of the PC-3-TxR and DU145-TxR cells to paclitaxel was performed at by estimating cell survival rates at different concentrations. In comparison to their parental cells, the IC50 of the PC-3-TxR cells was increased from 5.16 to 56.39 nM, and the IC50 of the DU145-TxR cells increased from 5.15 to more than 100 nM (Fig. 1A and Table 1). Perhaps the microenvironmental factor that has been demonstrated to have the largest influence on cells is heat. In order to assess the effects of heat exposure, the cell lines (resistant and sensitive) were exposed to 43°C for 1, 2, or 4 hours. After 48 hours of recovery, the survival rates of the paclitaxel resistant PC-3-TxR and DU145-TxR were compared with their parental cells; PC-3 and DU145, respectively. As a single modality the paclitaxel resistant PC-3-TxR and DU145-TxR cells showed a greater sensitivity to heat treatment. Compared to their parental cells, the IC50 of the resistant cell lines were decreased from 2.39 to 0.61 and 2.87 to 0.61 hours (Fig. 1B and Table 1). In addition to heat, the pH, UV and glucose play very important roles in the tumor microenvironment. The response of the resistant cell lines to these microenvironment stresses was also examined. In the pH stress experiments, PC-3-TxR and DU145-TxR cells exhibited a differential response to being exposed to higher pH values (1 value higher) for 48 hours, but not to lower pH values (1 value lower) (Fig 1C). The PC-3-TxR and DU145-TxR cell survival rates were not influenced significantly in comparison to their parent cells in response to UVC exposure. IC50 were from 16.39 to 11.74 and 16.34 to 14.36 seconds (Fig. 1D and Table 1). When cultured at low glucose concentration the paxitaxel-resistant cells had a lower survival rate in comparison to their parent cells. The IC50 increased from 0.19 to 0.86 mg/ml and 0.15 to 0.96 mg/ml (Fig. 1E and Table 1). The proliferation rates were examined in the PC-3 with PC-3-TxR and DU145 with DU145-TxR cells, but the data did not reveal significant differences (Fig. 3A and B). The results suggest that as cancer cells achieve resistance to chemotherapy they develop sensitivities to other types of microenvironmental stresses.

Fig. 1
Resistance to chemotherapy imparts a greater sensitivity to other microenviromental stresses. Evaluation of PC-3/TxR and DU145/TxR cell lines’ survival rates at different concentrations of the paclitaxel (A); exposure to 43°C heat for ...
Fig. 3
Time point proliferation assay in prostate cancer cell lines. A-D, PrEC, OPCT, LNCap, LN96, PC-3, PC-3-TxR, DU145 and DU145-TxR were cultured as described in materials and methods. The cells were seeded and counted different period in a hemocytometer. ...
TABLE I
IC50 Value of Prostate Cancer Cell Lines

Differential survival rates after exposure to various stresses in normal and cancer prostate cell lines

In order to study the evolution of prostate cancer resistance at each stage; primary cancer, metastasis, hormone-sensitive, hormone-refractory, chemotherapy-sensitive, and chemotherapy resistant, different cell models were utilized. As anticipated, the normal prostate epithelial (PrEC) cells had significantly higher survival rates when treated with paclitaxel as compared to the primary prostate cancer cells (OPCT). The IC50 in PrEC was over 100 nM, but in the OPCT cells was 1.01 nM (Fig. 2A, left and Table 1). The PrEC cells were also more viable in response to heat exposure longer than 2 hours (Fig. 2B, left), more stable at a higher pH value (Fig. 2C left) and more resistant to UV treatment (The IC50 was 15.43 seconds in PrEC, and 6.98 seconds in OPCT) (Fig. 2D, left and Table 1). The OPCT cells were less viable at low glucose concentrations (IC50 in PrEC was 1.50 mg/ml, but in OPCT was 1.01 mg/ml) (Fig. 2E, left and Table 1). In order to confirm that the differences in the response to glucose (Fig. 2E left) are not confounded by the use of DMEM medium for PrEC cells, we compared cell proliferation in PrEBM and DMEM media. There were no obvious differences (Fig. 3E). Furthermore, the hormone-therapy responsive cell, LNCap, was compared to the refractory line LN96. In contrast to LNCap, the LN96 cells were relatively more resistant to paclitaxel. The IC50 was 1.54 nM in the LNCap cells, but in LN96 was 4.41 nM (Fig. 2A, right and Table 1) and were significantly more viable in low glucose conditions. (IC50 was 0.48 mg/ml and 0.12 mg/ml respectively) (Fig. 2E, right and Table 1). However, the LN96 cells were more sensitive significantly to heat. The IC50 of LNCap was over 4 hours, but 3.44 hours in the LN96 (Fig. 2B, right and Table 1), high pH media (Fig. 2C, right) and UVC treatment (Fig 2D, right and Table 1). To ensure that differences in the proliferation capability did not influence these experiments, we compared the proliferation rates of these cells without treatment. The results suggest that there are no significant differences between the cell lines with the exception of slower proliferation of the PrECs compared to OPCT cells (Fig. 3C and D). To compare the observed phenotypes of all cells we estimated the IC50 values for paclitaxel and heat. The result summarized in Table 1 suggest that as prostate cancer progresses, from the primary cell line to paclitaxel resistant cell lines, together with the development of resistance to paclitaxel, an increased sensitivity to heat develops (Table 1). The ability of cancer cells to obtain resistant to therapies such as hormone deprivation, or chemotherapy appears to make them vulnerable to other types of stresses to which they were resistant before. Normal cells are much less sensitive to these stresses.

Fig. 2
Evaluation of PrEC, OPCT, LNCap and LN96 cell lines’ survival rates at different concentrations of the paclitaxel (A); exposure to 43°C heat for 0, 1, 2, and 4 hours (B); at different ± 1 pH values (C); exposure to UVC for 0, 15s, ...

Different apoptosis pathway (caspase3- dependent and independent) are utilized in cells exposed to distinct types of stresses

Cell apoptosis pathways are basic mechanisms in tissue homeostasis, cancer, and the induction of tolerance. As described above, the cell lines demonstrated differential viabilities in the context of different stress environments. Therefore, we next began to investigate the potential underlying apoptotic mechanisms of exposure to these district microenvironments. The effects of the stresses on individual apoptotic pathways in paclitaxel sensitive and paclitaxel resistance cell lines were examined. In the paclitaxel sensitive PC-3 and DU145 cells, after drug, or UV treatment, the apoptosis pathway in which caspase3 is involved appears to be activated, as opposed to the heat, basic pH and low glucose treatments (Fig. 4A and B left). In the paclitaxel resistant cell lines, PC-3-TxR and DU145-TxR, no evidence of apoptosis was observed after paclitaxel and UV treatment, whereas in PC-3 and DU145 (parental cells) evidence of caspase-3 induced apoptosis was present. However, after exposure to heat, or culturing at basic pH or in low glucose medium, we observed changes in PARP expression but not Caspase-3 (Fig. 4A and B right). These results suggest that in contrast to paclitaxel and UVC treatments, exposure to heat, high pH value, and low glucose may activate an alternative, caspase3-independent apoptosis pathway. Based on the apoptosis pathway results, the relationship between caspase3 induced apoptosis and non caspase-3 apoptotic pathways where different microenvironmental stresses were examined.

Fig. 4
Apoptosis pathways markers analysed in total protein from PC-3, PC-3-TxR, DU145 and DU145-TxR cell lines with (+) and without exposure (−) to different treatment (10 nM paclitaxel, 30s UVC, 2h 43°C Heat, +1 pH, and 1 mg/ml glucose).

Cells that survive and repopulate after exposure to heat and paclitaxel stress develop resistance to the same stresses but remain sensitive to others

To further investigate the phenotypic changes observed in cells as they develop resistance, the PC3 and DU145 (parental cell lines) were exposed to 100 nM of paclitaxel for 48 hours and then cultured at 43°C for 24 hours. After 30 days of recovery, the surviving cells generated singular colonies (Fig. 5A). The visualization of these cells by light microscopy showed distinct morphologic characteristics in comparison to their parental cells, and these cell lines were designated as PCAF48, DUAF48, PCAF24 and DUAF24 (Fig. 5B). The viability of the cell lines undergoing “acute” stress exposure (PCAF48 and DU145AF48) in comparison to those in which resistance was developed by “chronic” stress exposure (PC3-TxR and DU145-TxR), was compared along with the parental (PC3 and DU145) lines, by exposure to different concentrations of paclitaxel. The results indicate that the resistant cells developed by chronic exposure had the highest survival rates, the parental cells had the lowest and the cells that acutely developed resistance had intermediate survival rates, with the IC50 increasing from 5.16 to 10.01 nM in the PC-3 cells, and from 5.15 to 16.5 nM in the DU145 cells (Fig. 6A). As anticipated, the PCAF48 and DU145AF48 cells had lower survival rates when exposed to heat as compared to their parental cell lines (Fig. 6B). However, after two weeks of culture and four passages the “PCAF48-2” and “DU145AF48-2” demonstrated differential survival rates after exposure to paclitaxel or heat showing a trend toward restoration of the phenotype they had before exposure to the acute stresses (Fig. 6C and D). Similar effects were observed for PCAF24 and DUAF24, which appeared to be more resistant to heating and more sensitive to paclitaxel compared to their parental cell lines (Fig. 6E and F). And again, when retested after two weeks and 4 passages they tended toward restoration of their previous phenotype (Fig. 6G and H).

Fig. 5
Cell repopulation and modality after acute heat and paclitaxel treatment. A, PC-3 and DU145 cells treat with 24h heat or 48h 100 nM paclitaxel, after 30 days recovery, single clones are acquired. B, Microscope picture (x400) of the PC-3, DU145 (third ...
Fig. 6
The repopulated cells reappeared opposite response to heat and paclitaxel treatment and the response to heat and paclitaxel in deferent recovery time of repopulated cells. A, determined the paclitaxel resistance in PCAF48 and DUAF48 cell lines by cell ...

The above results suggest that the cancer cell population contains cells capable of surviving after acute extreme stress exposure by adaptive changes in their phenotype. At the same time the cells lose the ability to persist in the presence of other factors. However, after the acute stress factor has been withdrawn, the cells tend to restore the phenotype suited for better fitness and more efficient proliferation. These cells have more potent ability to survive and repopulate after acute stress exposure, could be the stem-like cells of the tumor. As CD133 is considered to be a tentative marker of the prostate cancer stem cell, we investigated expression of this surface molecule on the parental cells and the cells after repopulation following exposure to acute stress. The flow cytometry results that however only a small fraction from the repopulated cells have higher CD133 expression (Fig. 5C).

Overexpression of Na+K+ATPase and calcium binding protein in PC-3 and DU145 paclitaxel sensitive cells after heat treatement

Standard cancer therapies eradicate tumor cells at least in part by indirectly activating the apoptotic machinery. To investigate apoptotic pathways in both acute and chronic stress-induced resistant cell lines, cell extracts were obtained after 2 and 4 hours of exposure to heat or 10 nM concentrations of paclitaxel and apoptotic pathway component expression was revealed by immunoblot analysis. Similar to the previous experiments (Fig. 4), we observed caspase-3 independent apoptosis in the PC-3-TxR and DU145-TxR cells after 2 hours of heat that was more pronounced after 4 hours of heating (Fig. 7A). In this experiment we also investigated the expression of Na+K+ATPase and CALB1, which were found to be overexpressed in the parental but not in paclitaxel resistant cells after 2 and 4 hours of heating (Fig. 7A). This may indicate that these proteins play a role in resistance of the parental cells to heat, a feature that was lost after the cells developed paclitaxel resistance. Protein expression analysis of the cells after treatment with paclitaxel revealed Caspase-3 dependent apoptosis in the parental cells but no evidence of apoptosis was present in the resistant cells. The Na+K+ATPase and CALB1 were relatively overexpressed in the parental but down regulated in paclitaxel resistant cell lines (Fig. 5B). Similar protein expression was observed in the cell lines repopulated after acute stress exposure (Fig. 7C and D). These results suggest the same apoptosis pathways may work in both chronically and acutely resistance developed cell lines.

Fig. 7
After heat treatment, Na+K+ATPase and calcium binding protein CALB1 are overexpressed in paclitaxel sensitive PC-3 and DU145 but not in paclitaxel resistant PC-3-TxR, DU145-TxR PCAF48 and DUAF48 cell lines. A, total protein extracted from PC-3, PC-3-TxR, ...

The lethal effect of combination of heat with paclitaxel in prostate cancer cell lines

In order to study the synergistic killing effect of heat combine with paclitaxel in prostate cancer cell lines, we treated the cell lines with 43°C for 2 hours after exposure to 10 nM paclitaxel for 48 hours. When the cells were tested individually to paclitaxel, on the OPCT and LNCap cell lines, it demonstrated the significant killing effects, and no detectable effect on growth in PC-3-TxR, DU145-TxR, PCAF48 and DUAF48 cell lines in the presence of paclitaxel. Importantly the combination of heat with paclitaxel brings out a sensational synergistic lethal effect to all of the prostate cancer cell lines (Fig. 8A).

Fig. 8
Combination of Paclitaxel with Heat activited the cancer killing effect. A, the cell lines were either untreated or treated with paclitaxel (10 nM) for 48 hours, then plates had reached confluence and were stained with 0.5% typan blue. The paclitaxel ...

Discussion

In the current study, we report that resistance to chemotherapy imparts a greater sensitivity to microenvironmental stressors such as heat, basic pH and low glucose. As a control, normal prostate epithelial cells (PrEC) were shown to be much less sensitive to these stressful conditions. The collective finding is that the paclitaxel resistant prostate cancer cell lines, PC-3-TxR, DU145-TxR (stable) and PCAF48, DUAF48 (reversible), have gained resistance to chemotherapy but have in the process become hypersensitive to heat, basic pH and low glucose. Additionally, there are two types of apoptotic pathways invoked by stress treatments. Paclitaxel and UV stress treatments activated a caspase3-PARP apoptosis pathway in the paclitaxel sensitive cell lines, PC-3 and DU145, however, the other stresses, induced a caspase3-independent-PARP apoptotic pathway in the resistance cell lines, PC-3-TxR, DU145-TxR (stable) and. PCAF48, DUAF48 (reversible).

As we analyzed the spectrum of prostate cancer as represented by the models used, our findings demonstrate that resistance to one microenvironmental stress appears to result in sensitivity to others. Our study also indicates that prostate cancer cell lines develop resistance to paclitaxel gradually become sensitive to heat treatment. The inverse response between thermal- and chemotherapies suggest that combining stresses such as heat with currently used therapy may provide an opportunity for more efficacious use of these available approaches.

Since cancers are heterogeneous [Pardal et al., 2003], our analysis implicates two kinds of drug resistance that can be established in prostate cancer cell lines; one is a reversible drug resistance resulting from acute paclitaxel treatment, the other is a stable drug resistance caused by chronic paclitaxel treatment. An acute stress might spare tissue stem cells but injure other cell types. It has been demonstrated that in a chronic stress, there appears to be a more pronounced and ongoing influence on cell renewal systems with injury to all kinds of cell types, including stem cells [Johnstone and Baylin]. Considering reported links between drug resistance and a ‘cancer stem cell’ population, we examined CSC markers. The putative CSC marker CD133 was not highly expressed after long time recovery from the either acute or chronic exposures. The relationship between the reversibly drug resistant subpopulation and cancer stem cell is potentially complex. [Sharma et al.]. In the acute drug treatments, it was shown that drug resistant cancer cells behave similarly and that all of the tumor cells in a population potentially have the ability to stochastically acquire and relinquish this protective phenotype at a low frequency [Sharma et al.]. In the case of resistance after chronic drug treatment, tumor cells surviving have acquired genetic resistance due to secondary mutations [Dannenberg and Berns]. In the current study, both drug resistance (reversible and stable) cell lines developed by acute or chronic paclitaxel treatments respectively, showed an increased sensitivity to heat treatment. These results suggest that the repopulation maybe share a common characteristic by stress-induced, acute and chronic treatment, cellular adaptation. Regardless of which mechanism causes dug-resistance in vivo, thermal therapy may provide an operative way to treat drug-resistant prostate cancer.

Our findings also showed that after heat treatment, Na+K+ATPase and calcium binding protein CALB1 were up-regulated in both PC-3 and DU145 cell lines, thereby providing them with a mechanism to escape from this apoptotic pathway that is distinct from the PC-3-TxR and DU145-TxR cell lines. Cell ion channel proteins play an important role in cancer apoptosis, intracellular Ca2+ and Na+/K+ levels are involved in regulating the cell microenvironmental pH value, and in regulating the induction of apoptosis [Furuya et al., 1994; Mijatovic et al., 2009; Simpson et al., 2009]. The Na+/K+-ATPase helps maintain resting potential by active transport, and also functions as a signal transducer/integrator to regulate the MAPK pathway, reactive oxygen species (ROS), as well as intracellular calcium, and regulates cellular volume. For most animal cells it is responsible for 1/3 of the cell’s energy expenditure [Civan; Leaf, 1956; Wilson, 1954]. Furthermore, changes to the microenvironmental levels of ions such as Na+, K+ and Ca2+, may alter sensitivity to heat and chemotherapy. Implementation of stressors inducing alternative apoptosis pathways might be beneficial way to prevent the development of therapeutic resistance and post-treatment relapse of prostate cancer.

In summary, the ability of cancer cells to become resistant (reversible and stable) to therapies such as chemotherapy appears to make them vulnerable to stresses in their environment; however, normal cells are much less sensitive to stressful conditions such as heat. In lieu of current therapy, increasing drug resistance causes an increased sensitivity to heat. Therefore combining stresses such as heat with currently used therapies may provide an opportunity for the more efficacious use of these available approaches (Fig. 8B).

Recently new non-invasive nanoparticle-mediated approaches were reported opening new possibilities of application of different treatments in combination [Chopra et al., 2009; Coffey et al., 2006; Krishnan et al.]. Future therapeutic strategies will undoubtly focus on integrating microenvironmental stresses with current chemotherapy and radiation [McTaggart and Dupuy, 2007; O’Neal et al., 2004]. All thermal therapies offer the advantage of flexible, low cost treatment approaches through percutaneous, laparoscopic, and open surgical access. Thermal therapies are also minimally invasive outpatient procedures with repeatability and reproducibility of results between patients [Carrafiello et al., 2008]. Taken together, thermal therapy may be a cost effective approach for the treatment of chemotherapy resistant prostate cancer. Accumulating evidence indicates that treatment plays a very important role in cancer cell evolution [Becker et al., 2000; Collingridge et al., 1999; Vaupel et al., 2001; Vaupel et al., 2003].

Acknowledgments

We would like to thank David Yeater and Don Vindivich for their technical assistance. This work was supported by grant U54CA143803 from NCI.

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