|Year : 2012 | Volume
| Issue : 6 | Page : 714-721
Effect of lithium chloride and antineoplastic drugs on survival and cell cycle of androgen-dependent prostate cancer LNCap cells
Vajihe Azimian-Zavareh1, Ghamartaj Hossein2, Ehsan Janzamin3
1 Department of Animal Biology, Developmental Biology Lab., School of Biology, University College of Science, University of Tehran; Department of Regenerative Medicine, Royan Institute for Stem Cell Biology and Technology, Tehran, Iran
2 Department of Animal Biology, Developmental Biology Lab., School of Biology, University College of Science, University of Tehran, Iran
3 Department of Stem Cells, Royan Institute, Tehran, Iran
|Date of Submission||25-Nov-2011|
|Date of Decision||31-Aug-2012|
|Date of Acceptance||01-Sep-2012|
|Date of Web Publication||8-Nov-2012|
Department of Animal Biology, Developmental Biology Lab., School of Biology, University College of Science, University of Tehran
Source of Support: This work was partly supported by Grant # 26830/6/010 from University College of Science, University of Tehran, Conflict of Interest: None
Objective: Glycogen synthase kinase-3β (GSK-3β) has been reported to be required for androgen receptor (AR) activity. This study sought to determine the usefulness of lithium chloride (LiCl) as a highly selective inhibitor of GSK-3β to increase the sensitivity of LNCap cells to doxorubicin (Dox), etoposide (Eto), and vinblastine (Vin) drugs.
Materials and Methods: Thiazolyl Blue Tetrazolium Blue (MTT) assay was used to determine the cytotoxic effect to LiCl alone or in combination with low dose and IC 50 doses of drugs. Subsequently, cell cycle analysis was performed by using flow cytometry.
Results: LiCl showed cytotoxic effect in a dose- and time-dependent manner (P<0.001). Both Dox (100 or 280 nM) and Vin IC 50 (5 nM) doses caused G2/M-phase arrest (P<0.001) compared with control. However, low dose (10 μM) or IC 50 (70 μM) Eto doses showed G2/M or S-phase arrests, respectively (P<0.001). Combination of low dose or IC 50 dose of Eto with LiCl showed increased apoptosis as revealed by high percent of cells in SubG1 (P<0.05, P<0.01, respectively). Moreover, Eto (10 μM) led to decreased percent of cells in G2/M phase when combined with LiCl (P<0.05).
Conclusion: This study showed that LiCl increases apoptosis of (LNCap) Lymph Node Carcinoma of the Prostate cells in the presence of Eto, which is S- and G2-phase-specific drug.
Keywords: Antineoplastic drugs, combination therapy, glycogen synthase kinase-3β, lithium chloride, prostate cancer
|How to cite this article:|
Azimian-Zavareh V, Hossein G, Janzamin E. Effect of lithium chloride and antineoplastic drugs on survival and cell cycle of androgen-dependent prostate cancer LNCap cells. Indian J Pharmacol 2012;44:714-21
|How to cite this URL:|
Azimian-Zavareh V, Hossein G, Janzamin E. Effect of lithium chloride and antineoplastic drugs on survival and cell cycle of androgen-dependent prostate cancer LNCap cells. Indian J Pharmacol [serial online] 2012 [cited 2021 Aug 2];44:714-21. Available from: https://www.ijp-online.com/text.asp?2012/44/6/714/103265
| » Introduction|| |
Prostate cancer (PCa) remains second most common cancer among men in the world.  Unfortunately, there is no effective cure for PCa once cancer has progressed from androgen-dependent state to a hormone refractory state, and molecular basis for this phenomenon is largely unknown.
Several different molecular mechanisms might be responsible for the transition to androgen independence. Many of these involve the mutations in the androgen receptor (AR) and the altered AR co-regulator signaling,  but they might also include enhanced expression of growth factor receptors and their associated ligands.  It is known that androgens influence growth by shortening the length of G1/G0 leading to accelerating entry into S phase.  AR regulates cellular proliferation by control of cyclin-dependent kinase (CDK) and cyclins at the transcriptional level and by posttranslational modifications that influence cell cycle protein activity.  These observations point to a direct role of AR in progression of AR-positive PCa cells from G1 into S phase by its participation in the events leading to the assembly and/or function of DNA replication machinery.  Therefore, some of the enzymes and proteins interacting with AR in DNA replication machinery may prove to be ideal targets, in combination with androgen-ablation for an effective treatment of androgen sensitive, as well as androgen-refractory PCa.  This includes modulation of signal transduction pathways, which may delay PCa progression.
Recent studies have shown that glycogen synthase kinase-3β (GSK-3β) is involved in many biological processes, including cell cycle progression, gene transcription, apoptosis/survival, cellular metabolism, cell movement, tumorigenesis, cytokinesis, and embryonic development, although no mutation in the GSK-3β gene was found in human cancers. 
Recently, the role of GSK-3β in certain PCa cell lines has been investigated in vitro showing the requirement of GSK-3β activity in AR transactivation in androgen-responsive PCa cells.  Accordingly, other study showed that GSK-3β inhibitors reduced the growth of PCa cell in vitro in AR-expressing cell lines. Moreover, GSK-3β inhibitor SB216763 did not affect growth in AR-null PC-3 cells, and it was concluded that GSK-3β-induced proliferative effect is directly mediated via its interaction with AR.  Controversy still exists about the role of GSK-3β in cancer progression, as other groups showed suppressive effect of GSK-3β on AR transactivation. ,
In an in vitro cell culture model, it was found that GSK-3β inhibitors, such as lithium chloride (LiCl), suppress cancer cell growth, induce S-phase cell cycle arrest, and abolish DNA replication in a time- and dose-dependent manner.  Moreover, the suppressive effects of LiCl on PCa cells were determined to be associated with downregulation of DNA replication-related genes including cdc6, cyclin A and E, as well as cdc25C and upregulation of CDK inhibitor p21 CIP1.  In addition, a significant inverse relationship was shown between cancer development and LiCl dose  as LiCl and other specific GSK-3β inhibitors were found to significantly suppress tumor growth in a mouse xenograft model without any appreciable side effects.  Recent study reported that high levels of activated GSK-3β known as pGSK-3βY 216 were associated with aggressive PCa  and are a critical determinant in the progression of PCa. 
Cytotoxic chemotherapy is being used to control and treat PCa but remains relatively nonselective and highly toxic to normal tissues. In an effort to develop effective strategies that increase the therapeutic potential of cytotoxic anticancer drugs with less systemic toxicity in recent years, more efforts are being directed toward combination chemotherapy.  In this regard, dietary supplements with high anticancer efficacy and least toxicity to normal tissues are suggested as possible candidates to be investigated for their synergistic efficacy in combination with anticancer drugs. It is anticipated that the PCa cells arrested in S phase will be more sensitive to other cytotoxic drugs , ; as LiCl induced S-phase arrest in PCa cell lines,  this promoted us to use it in combination with antineoplastic drugs. In this study, we assess the cytotoxic effect of three antineoplastic drugs with different mechanism of action in combination with LiCl on androgen-dependent LNCaP cell line. The anthracycline antibiotic doxorubicin (Dox) is a cell cycle nonspecific drug, which may cause cell cycle arrest in different cell cycle phase. However, etoposide (Eto) is a semisynthetic derivative of the podophyllotoxins, which inhibits DNA synthesis by inhibiting DNA topoisomerase II. Eto is a cell cycle dependent and phase specific, affecting mainly the S and G 2 phases. Vinblastin (Vin) is a vinca alkaloid which binds tubulin, thereby inhibiting the assembly of microtubules and is M-phase cell cycle specific agent.  The aims of this study were threefolds: (1) to assess the sensitivity of LNCap cells to LiCl, (2) as LNCap have been reported to be resistant to Dox and Eto,  we sought to determine whether the cytotoxic effects of Dox and Eto on these cells would be modulated in combination with LiCl, and (3) whether cell cycle specificity of drugs may be a determinant factor for their selection in combination therapy with LiCl.
| » Materials and Methods|| |
Cell Lines and Reagents
Human prostate carcinoma LNCaP cells were obtained from Pasteur Institute of Iran and grown in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum and antibiotics at 37°C in a 5% CO 2 atmosphere under 90-95% humidity. LiCl and sodium chloride (NaCl) were obtained from Merck (Darmstadt, Germany), and 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) and propidium iodide were obtained from Sigma-Aldrich (Saint Louis, USA). RNase A was purchased from iNtRON Biotechnology (Seoul, Korea). Antineoplastic drugs were obtained from Iranian Red Cross Pharmacy.
Cytotoxicity and Anticancer Assay
The IC 50 of lithium and drugs on LNCap cells was measured by MTT-based cell proliferation assay, which was calculated by using linear regression test of Graphpad Instate-3 software (LaJolla, CA, USA). To this order, LNCaP (10,000 cells/well) cells were seeded in 96-well plates and next day were treated with either RPMI 1640 alone as a control or with different doses of LiCl (2.5, 10, 25, and 45 mM) LNCap cells. In parallel, cells were treated with Dox (0, 5, 10, 25, 100, 250, 500 nM), Eto (0, 2.5, 5, 10, 50 μM), and Vin (0, 0.25, 0.5, 1, 2.5, 5, 10 nM) alone, or in combination with LiCl for 24, 48, and 72 h. At the end of exposure, 10 μL of MTT (Sigma-Aldrich, Saint Louis, USA) stock solution (5 mg/mL) was added to 100 μL of medium in each well, and plates were incubated for 4 h at 37°C, and subsequently, MTT-formazan product was dissolved by adding 100 μL of acidic isopropanol to each well. The plates were incubated about 10 min at room temperature before determination of absorbance (A) values at 570 nm with Elisa reader (Awarness Technology Inc., Palm City, FL., USA). Cell survival was expressed as percentage (A value of treated to control cells ratio×100). Experiments were performed in triplicate and repeated at least three times. To rule out the effect of the anion (Cl− ) through an osmotic disturbance, we compared LiCl with NaCl at equimolar concentrations.
Flow Cytometry Assay
Cell cycle distribution and measurement were performed by flow cytometry. To this order, cells (5×10 5 cell/ml) were seeded and the day after were serum-starved for 24 h then returned to serum-containing media with LiCl and drug either alone or in combination for 48 h. After treatment, pool of floating cells in the medium and harvested attached cells were washed with cold phosphate-buffered saline (PBS) and fixed in 70% ethanol (-20°C) at 4°C. After 2 h, fixed cells were pelleted and stained with propidium iodide (20 μg/ml) in the presence of RNase A (100 μg/ml) for 30 min at 37°C, and about 10 4 cells were analyzed using a fluorescence-activated cell sorter. Data were analyzed by using WinMDI 2.9 software.
Nominal variables were analyzed by the use of the Kolmogorov-Smirnov test. Skewed and normal distributed metric variables were analyzed by Mann-Whitney U and one-way ANOVA tests, respectively. Analysis of combined growth-inhibitory effect was performed by using two-way ANOVA. P≤0.05 was considered statistically significant. All statistical procedures were run on SPSS version 16 (SPSS Inc., Chicago, IL, USA).
| » Results|| |
LiCl Effect on LNCap Cell Growth
LNCap cells viability in the presence or absence of LiCl (2.5-45 mM) was assessed as percent of viable cells compared with control (in the absence of LiCl). Cells showed 23% (P <0 0.001) reduced cell viability with 45-mM LiCl after 24 h [Figure 1]a. Comparable cell viability was obtained with 25-mM (P <0 0.01) and 47% reduction with 45-mM (P <0 0.001) LiCl after 48 h. The sensitivity of LNCap cells to LiCl was time dependent as significant decreased cell viability was observed with low as well as high dose of LiCl after 72 h [Figure 1]a. No significant effect was observed with NaCl (2.5-45 mM) as control [Figure 1]b. In the next step, to define the usefulness of antineoplastic drugs and LiCl combination therapy, cytotoxic assay was performed with drugs alone and in combination with LiCl.
|Figure 1: Effect of LiCl (a) and NaCl (b) on viability of LNCap cells. Values are expressed as mean+SD from at least three independents experiments in triplicate. *P<0.05, **P<0.01, ***P<0.001 compared with control|
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Dose-response curves for each drug are shown in [Figure 2]. Dox had no cytotoxic effect on LNCap cells after 24 h and showed a significant cytotoxic effect after 48 and 72 h with high dose of 250 (P <0 0.01) and 500 nM (P <0 0.001), respectively [Figure 2]a. Eto had no effect on LNCap cells after 24 h; however, cell viability reduction was observed with 10-100 nM after 48 (P <0 0.05) and 72 (P <0 0.01) h [Figure 2]b. However, Vin showed a strong and significant cytotoxic effect on LNCap cells [Figure 2]c.
|Figure 2: Effect of LiCl and antineoplastic drugs alone or in combination on LNCap cell growth. (a) Doxorubicin (Dox), (b) etoposide (Eto), and (c) vinblastine (Vin). Values are expressed as mean+SEM relative to control.*P<0.05, **P<0.01, ***P<0.001|
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Effect of LiCl and Antineoplastic Drugs in Combination on LNCap Cells Growth
Next, we sought to determine whether antineoplastic drugs in combination with LiCl may increase the sensitivity of these cells to these drugs. Based on obtained results with different timing, the selected time for these experiments was 48 h as a best time for assessment of combination therapy on cells viability. Two different doses of drugs (1) IC 50 dose and (2) low dose of each drug alone or in combination with low dose (20 mM) and IC 50 dose (45 mM) of LiCl were used.
For combination effect of Dox and LiCl [Figure 3]a, concentration of 100 nM led to 22% decreased cell viability, and showed 33% reduction in cell viability when combined with 20-mM LiCl (P <0 0.01 compared with control). This result was comparable with 20-mM LiCl alone. However, 100-nM Dox+45-mM LiCl showed 49.6% cell viability reduction (P <0 0.001 compared with control, P <0 0.01 compared with 100 nM). Treatment with Dox IC 50 dose (280 nM) alone showed 46.5% cell viability reduction (P <0 0.001), which was most effective in combination with 20-mM LiCl (55% reduction, P <0 0.001 compared with control; P <0 0.05 compared with LiCl alone) as well as with 45-mM LiCl (68% reduction; P <0 0.001 compared with control, P <0 0.01 compared with 280-nM and 45-mM LiCl alone). Thus, the use of IC 50 doses of Dox and LiCl in combination was more effective and induced a highly significant cytotoxic effect compared with each substance alone [Figure 3]a.
|Figure 3: Dose-response curves of LNCap cells after 24, 48, and 72 h of exposure to (a) doxorubicin, (b) etoposide, and (c) vinblastine. Values are expressed as mean±SEM from at least three independent experiments in triplicate. IC 50 values were calculated by using instat-3 software. aP<0.05, bP<0.01, cP<0.001 compared with control|
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For combination effect of Eto and LiCl, a low dose (10 μM) and IC 50 dose (70 μM) of Eto alone or in combination with LiCl was used. As shown in [Figure 3]b, 10-μM Eto did not showed significant effect on cells viability compared with control, but in combination with two concentration of LiCl (20 and 45 mM), a significant reduction of cells viability was observed compared with control (30% P <0 0.05; and 46% P <0 0.01, respectively). Also, this reduction of cells viability in co-treatment of 45-mM LiCl with 10-μM Eto was significant relative to 10- μM Eto alone (26%, P <0 0.05). Treatment of cells with 70-μM Eto alone showed 48% cell growth inhibition (P <0 0.01), and combination of this dose with 20-mM LiCl showed significant cell growth inhibition compared with control and 20-mM LiCl alone (51% P <0 0.05 and P <0 0.05, respectively). Although, the IC 50 dose of Eto showed significant cytotoxic effect on cells however, its combination with 45-mM LiCl revealed a highly significant cell growth inhibitory effect (62% compared with control P <0 0.001; compared with Eto or LiCl alone P <0 0.05). Altogether, these results showed that a nonsignificant cytotoxic effect with low dose of Eto could be significantly effective in combination with LiCl, which was more effective by using IC 50 dose of Eto with LiCl [Figure 3]b.
As it has been shown in [Figure 3]c, Vin had a high cytotoxic effect on LNCap cells; thus, a low dose of 25 nM was used as low dose, which showed 20% cell viability reduction although not significant. However, IC 50 dose (5 nM) caused 51% reduced cell viability (P<0.001). The combined effect of 5-nM Vin plus 45-mM LiCl led to 60% reduced cell viability, which was highly significant compared with control (P<0.01) and LiCl alone (P<0.05). It should be noted that there was no significant difference on cell growth inhibition between 1 and 5 nM concentrations (49% and 51%, P<0.001, respectively). However, the combined effect of 1-nM Vin plus 45-mM LiCl led to 64% reduced cell viability, which was highly significant compared with control (P<0.001) and drugs alone (P<0.01 compared with Vin vs. P<0.05 compared with LiCl) (data not shown).
Effect of LiCl-Antineoplastic Drugs Combination on Cell Cycle Progression of LNCap Cells
To determine if combination of drug and LiCl-induced inhibition of proliferation of LNCap cells was due to altered cell cycle regulation, LNCap cells were serum-starved for 24 h and then treated with LiCl and drugs alone or in combination for 48 h. Cell cycle profiles were monitored by flow cytometric analysis of DNA content in the absence or presence of IC 50 and low doses of each drug alone or in combination with low (20 mM) and IC 50 doses (45 mM) of LiCl.
Treatment of cells with different concentrations of LiCl showed an increase of cell percentage in S and G2/M phase, although not significant [Figure 4], [Table 1]. Different doses of Dox caused significant decrease of cells proportion in G1 and G2/M phases [Figure 4], [Table 1]. Cell cycle analysis for Dox alone or in combination did not show a significant change in proportion of cells in SubG1 [Figure 4], [Table 1]. Cell proportion in G1 phase was decreased significantly with Dox 100 nM+20-mM or 45-mM LiCl compared with control [Figure 4], [Table 1]. [Table 1] shows G1 arrest with Dox 280 nM+LiCl 45-mM compared with IC 50 dose of Dox alone (P <0 0.001). S-phase arrest was significant with 100-nM Dox+45-mM LiCl and 280-nM Dox combined with low or high concentrations of LiCl [Figure 4], [Table 1]. The percentage of cells in G2/M phase was increased with Dox in combination with 20-mM LiCl [Figure 4], [Table 1]; however, when combined with 45-mM LiCl, cell proportion in G2/M phase was decreased compared with Dox alone [Figure 4], [Table 1].
|Figure 4: Cell cycle analysis of LNCap cells in the presence of LiCl or Dox alone or in combination by flow cytometry. 280 nM and 100 nM represent IC 50 and low dose, respectively. 45-mM and 20-mM LiCl represents IC 50 and low dose, respectively. M1: subG1, M2: G1, M3: S, M4: G2/M phases. Figure is representative of one of three independent experiments|
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|Table 1: Effect of Dox and LiCl alone or in combination on cell cycle progression of LNCap cells|
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Low or IC 50 dose of Eto caused significant G2/M and S arrests, respectively [Figure 5], [Table 2]. It should be noted that IC 50 or low doses of Eto in combination with IC 50 dose of LiCl showed a significant increase in proportion of cells in SubG1 [Figure 5], [Table 2]. Percent of cells in G1 phase decreased significantly with Eto 10 μM+20-mM or 45-mM LiCl compared with control [Figure 5], [Table 2]. A significant G1-phase arrest (P<0.01) with Eto 70 μM+LiCl 45-mM compared with IC 50 dose of Eto alone was observed, although proportion of cells in G1 was significantly lower compared with control [Table 2]. S-phase arrest was only observed with 10 μM Eto+20-mM LiCl or Eto 70 μM alone [Figure 5], [Table 2]. Interestingly, the proportion of cells in G2/M phase was significantly (P<0.001) increased with 10-μM Eto alone, which was higher compared with Eto 10 μM+LiCl (20 or 45 mM) [Figure 5], [Table 2].
|Figure 5: Cell cycle analysis of LNCap cells in the presence of LiCl or Eto in combination by flow cytometry. 70 mM and 10 mM represent IC 50 and low dose, respectively. 45-mM and 20-mM LiCl represents IC 50 and low dose, respectively. M1: subG1, M2: G1, M3: S, M4: G2/M phases. Control and LiCl alone were shown in Figure 4. This figure is representative of one of three independent experiments|
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|Table 2: Effect of Eto and LiCl alone or in combination on cell cycle progression of LNCap cells|
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Vin led to a higher proportion of cells in SubG1 (P<0.001) compared with control, whereas this effect was lesser in combination with LiCl [Figure 6], [Table 3]. Low dose of Vin had no influence on G1 phase; however, when combined with 20- or 45-mM LiCl, there was a decrease in percent of cells in G1 phase (P<0.05; P<0.001, respectively) [Figure 6], [Table 3]. Although IC 50 dose (Vin 5 nM) alone or in combination with 20-mM LiCl showed a significant 6.4-fold decrease of cell proportion in G1 phase (P<0.001), combination with 45-mM LiCl led to 2.5-fold decrease compared with control (P<0.001) [Figure 6], [Table 3]. This means that the percent of cells in G1 with Vin 5 nM+LiCl 45-mM was significantly (P<0.001) increased compared with IC 50 dose of Vin alone [Table 3]. No significant change of cells proportion in S phase was observed [Figure 6], [Table 3]. Compared with control, a 3.0-fold increase of cells in G2/M phase was observed with IC 50 dose of Vin, which was comparable when combined with 20-mM LiCl (P<0.001); however, combination with 45-mM LiCl attenuated this effect (2.2-fold increase compared with control, P<0.001) [Figure 6], [Table 3]. It should be noted that despite a little difference in G2/M percent of cells between Vin 5 nM alone or in combination with LiCl 45 mM, this change remained significant (P<0.01) [Table 3].
|Figure 6: Cell cycle analysis of LNCap cells in the presence of LiCl or Vin in combination by flow cytometry. 5 nM and .05 nM represent IC 50 and low dose, respectively. 45-mM and 20-mM LiCl represent IC 50 and low dose, respectively. M1: subG1, M2: G1, M3: S, M4: G2/M phases. Control and LiCl alone were shown in Figure 4. This figure is representative of one of three independent experiments|
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|Table 3: Effect of Vin and LiCl alone or in combination on cell cycle progression of LNCap cells|
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| » Discussion|| |
The goal of this study was to assess the usefulness of LiCl as an inhibitor of GSK-3β enzyme in combination therapy with antineoplastic drugs to increase the cytotoxic effect of these drugs on LNCap cell line. GSK-3β is able to regulate many signaling pathways because it possesses unique structural characteristics that cause it to recognize prephosphorylated substrates.  Moreover, GSK-3β and AR codistribute in immunohistochemical staining of PCa tissue samples, which showed that GSK-3β and AR were diffusely distributed in the cytoplasm or concentrated in the nuclei of PCa cells.  Meanwhile, cytoplasmic accumulation of GSK-3β protein in prostate cancers was found to correlate with disease progression.  Furthermore, aberrant GSK-3β activation (Y216 phosphorylation) was shown in highly aggressive PCa cells.  Consistently, suppressing GSK-3β activity reduced PCa cell proliferation in vitro.,,
The codistribution and also coimmunoprecipitation of GSK-3β and AR in PCa tissue suggest that these molecules are capable of interacting in both the cytoplasmic or nuclear compartments of PCa cells, suggesting that these two molecules may interact during the progression of PCa. Indeed, both activated or inhibited form of GSK-3β phosphorylation on Y216 or serine 9 detected in LNCap cells, respectively. ,
However, controversy may still exist on the role of GSK-3β on AR activity, as one study showed active GSK-3β is required for inhibition of AR activity.  While other studies support firm evidences for the role of GSK-3β activity as a required enzyme for AR activity. ,, In addition, it has been shown that AR is necessary for G1 to S transition,  and previous report showed that LiCl caused S-phase arrest of LNCap cells by reduction of E2F-mediated gene expression and reduced cdc25C expression, a protein phosphatase required for S-phase completion and mitosis entry.  In agreement with previous reports, this study showed the antiproliferative effect of LiCl on LNCap cells. The emergence of resistant clones is a universal problem of chemotherapy. However, it seems that its most acute manifestation is the failure to treat metastasis. A part of this problem is the imperfect effectiveness of adjuvant chemotherapy as the tool to eradicate undetectable micro metastasis. In view of toxicity of anticancer drugs, optimal scheduling is potentially useful in improving these treatments. One of the conceivable strategies of protocol optimization, exploiting drug specificity, is to arrest cancer cells in the S phase.  As shown in the present study, LiCl may cause S-phase arrest in LNCap cells thus, it may be a useful therapeutic tool in PCa. LNCap cells in the presence of LiCl [Table 1], which may be a useful therapeutic tool as it causes S-phase arrest of these cells.
As mentioned above, GSK-3β has multiple substrates; many of them are involved in cell cycle regulation. It is well known that phosphorylation of cyclin D1 by GSK-3β led to ubiquitination and degradation of this cyclin by proteasomes.  Interestingly, cyclin D1 plays a negative feedback loop as its increased levels have a negative effect on AR activity.  Thus, it is tempting to speculate that inactivation of GSK-3β by LiCl may increase intracellular cyclin D1 levels and subsequent inhibition of AR activity. Moreover, other study showed increased P53 stabilization, which was also observed in our study (data not shown), and increased levels of P21 in the presence of LiCl. 
This study for the first time demonstrates the usefulness of LiCl and antineoplastic drugs in combination to increase cytotoxic effect of chemotherapeutic drugs on an androgen-dependent PCa cell line. We showed that despite resistance of LNCap cells to Dox when combined with LiCl, the percent of cells in SubG1 and also in G1 phases increased and reduced number of cells in G2/M phase; thus, it prevents cell cycle progression [Figure 4], [Table 1]. As Dox has serious side effects, any agent that may lead to more effective cytotoxic effect or use of lower dose of this agent may be an interesting candidate for combination therapy. The other finding of this study was combination of LiCl with antineoplastic agents affecting microtubules organization leading to G2/M arrest may not increase effectiveness of this kind of drug [Figure 5], [Table 3]. Here, we demonstrated that use of Vin as an antimitotic drug with LiCl led to reduced cytotoxic effect of Vin as the percent of cells in SubG1 was reduced [Figure 6], [Table 3]. The most interesting finding of this study is the significant increase of apoptotic cells (increased percent of cells in SubG1) when Eto was used in combination with LiCl. Of note, Eto is a cell cycle-specific agent affecting mainly S and G2 phases, as LiCl alters cell cycle by causing S- and G2-phase arrest; thus, use of these drugs together showed a greater additive cytotoxic effect [Figure 5], [Table 2]. Altogether, these results suggest that the choice of antineoplastic drugs based on their cell cycle-specific effect should be considered when used in combination with LiCl.
In support of our study for usefulness of LiCl in combination therapy, a very recent study showed that LiCl significantly suppressed tumor development and growth of subcutaneous xenografts derived from human PCa cells.  This study revealed that following GSK-3β inhibition, CCAAT/Enhancer Binding Protein alpha (C/EBP alpha), a negative cell cycle regulator, was remarkably accumulated in xenograft tumors or in cultured PCa cells and knocking down C/EBPa expression abolished GSK-3β inhibition-induced suppression of E2F1 transactivation, suggesting that C/EBPa accumulation is involved in GSK-3β inhibition-induced antitumor effect. 
Of interest, LNCap cells are phosphatase and tensin homolog (PTEN) null, which means constitutive activation of PI3K/Akt survival pathway.  As Akt/PKB leads to GSK-3β inactivation, this may explain the higher levels of pGSK3bSer 9 in LNCap cells compared with other PCa cell lines.  However, our results clearly showed that even in the PTEN null cells, LiCl modulates AR activity, and this may indicate the needs for further investigation to elucidate the mechanism of LiCl effect on AR activity.
Taken together, LiCl significantly reduced cancer incidence compared with the controls both in clinical observation and animal studies, indicating its possible value in human cancer intervention. ,,,,,,,, This study for the first time demonstrates LiCl as a potential molecule in combination therapy in an in vitro model of androgen-dependent prostate cancer cell line. Finally, it should be noticed that the choice of antineoplastic drugs based on their cell cycle specificity and/or mechanism of action in addition to PCa cell type depending on their hormone responsiveness and/or harbored mutation of key proteins involved in cell cycle regulation may influence the rational for the use of LiCl in combination therapy.
| » Acknowledgments|| |
We are grateful to Dr. M. Malek for helpful advices regarding statistical analysis from School of Biology, University College of Science, and University of Tehran. This work was partly supported by Grant # 26830/6/010 from University College of Science, University of Tehran.
| » References|| |
|1.||Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin 2005;55:74-108. |
|2.||Fujimoto N, Yeh S, Kang HY, Inui S, Chang HC, Mizokami A, et al. Cloning and characterization of androgen receptor coactivator, ARA55, in human prostate. J Biol Chem 1999;274:8316-21. |
|3.||Yeh S, Lin HK, Kang HY, Thin TH, Lin MF, Chang C. From HER2/Neu signal cascade to androgen receptor and its coactivators: A novel pathway by induction of androgen target genes through MAP kinase in prostate cancer cells. Proc Natl Acad Sci U S A 1999;96:5458-63. |
|4.||Culig Z, Hobisch A, Bartsch G, Klocker H. Androgen receptor-an update of mechanisms of action in prostate cancer. Urol Res 2000;28:211-9. |
|5.||Sivanandam A, Murthy S, Kim SH, Barrack ER, Veer Reddy GP. Role of androgen receptor in prostate cancer cell cycle regulation: Interaction with cell cycle regulatory proteins and enzymes of DNA synthesis. Curr Protein Pept Sci 2010;11:451-8. |
|6.||Kim L, Kimmel AR. GSK3, a master switch regulating cell-fate specification and tumorigenesis. Curr Opin Genet Dev 2000;10:508-14. |
|7.||Liao X, Thrasher JB, Holzbeierlein J, Stanley S, Li B. Glycogen synthase kinase-3beta activity is required for androgen-stimulated gene expression in prostate cancer. Endocrinology 2004;145:2941-9. |
|8.||Mazor M, Kawano Y, Zhu H, Waxman J, Kypta RM. Inhibition of glycogen synthase kinase-3 represses androgen receptor activity and prostate cancer cell growth. Oncogene 2004;23:7882-92. |
|9.||Wang L, Lin HK, Hu YC, Xie S, Yang L, Chang C. Suppression of androgen receptor-mediated transactivation and cell growth by the glycogen synthase kinase 3 beta in prostate cells. J Biol Chem 2004;279:32444-52. |
|10.||Salas TR, Kim J, Vakar-Lopez F, Sabichi AL, Troncoso P, Jenster G, et al. Glycogen synthase kinase-3 beta is involved in the phosphorylation and suppression of androgen receptor activity. J Biol Chem 2004;279:19191-200. |
|11.||Sun A, Shanmugam I, Song J, Terranova PF, Thrasher JB, Li B. Lithium suppresses cell proliferation by interrupting E2F-DNA interaction and subsequently reducing S-phase gene expression in prostate cancer. Prostate 2007;67:976-88. |
|12.||Cohen P, Goedert M. GSK3 inhibitors: Development and therapeutic potential. Nat Rev Drug Discov 2004;3:479-87. |
|13.||Zhu Q, Yang J, Han S, Liu J, Holzbeierlein J, Thrasher JB, et al. Suppression of glycogen synthase kinase 3 activity reduces tumor growth of prostate cancer in vivo. Prostate 2011;71:835-45. |
|14.||Liao X, Thrasher JB, Holzbeierlein J, Stanley S, Li B. Glycogen synthase kinase-3beta activity is required for androgen-stimulated gene expression in prostate cancer. Endocrinology 2004;145:2941-9. |
|15.||Chen CD, Welsbie DS, Tran C, Baek SH, Chen R, Vessella R, et al. Molecular determinants of resistance to antiandrogen therapy. Nat Med 2004;10:33-9. |
|16.||Figg WD, Arlen P, Gulley J, Fernandez P, Noone M, Fedenko K, et al. A randomized phase II trial of docetaxel (taxotere) plus thalidomide in androgen-independent prostate cancer. Semin Oncol 2001;28:62-6. |
|17.||Balk SP, Knudsen KE. AR, the cell cycle, and prostate cancer. Nucl Recept Signal 2008;6:e001. |
|18.||Roy S, Kaur M, Agarwal C, Tecklenburg M, Sclafani RA, Agarwal R. p21 and p27 induction by silibinin is essential for its cell cycle arrest effect in prostate carcinoma cells. Mol Cancer Ther 2007;6:2696-707. |
|19.||Dorr RT, Von-Hoff DD. Drug monographs. Cancer Chemotherapy Handbook. 2 nd ed. Norwalk, Conneticut: Appleton and Lange; 1994. p. 395-416. |
|20.||van Brussel JP, van Steenbrugge GJ, Romijn JC, Schröder FH, Mickisch GH. Chemosensitivity of prostate cancer cell lines and expression of multidrug resistance-related proteins. Eur J Cancer 1999;35:664-71. |
|21.||Li R, Erdamar S, Dai H, Sayeeduddin M, Frolov A, Wheeler TM, et al. Cytoplasmic accumulation of glycogen synthase kinase-3beta is associated with aggressive clinicopathological features in human prostate cancer. Anticancer Res 2009;29:2077-81. |
|22.||Rinnab L, Schütz SV, Diesch J, Schmid E, Küfer R, Hautmann RE, et al. Inhibition of glycogen synthase kinase-3 in androgen-responsive prostate cancer cell lines: Are GSK inhibitors therapeutically useful? Neoplasia 2008;10:624-34. |
|23.||Brown BW, Thompson JR. A rationale for synchrony strategies in chemotherapy. In: Ludwig C, editor. Epidemiology. Philadelphia: SIAM Publication; 1975. p. 31-48. |
|24.||Takahashi-Yanaga F, Sasaguri T. GSK-3beta regulates cyclin D1 expression: A new target for chemotherapy. Cell Signal 2008;20:581-9. |
|25.||Davies MA, Koul D, Dhesi H, Berman R, McDonnell TJ, McConkey D, et al. Regulation of Akt/PKB activity, cellular growth, and apoptosis in prostate carcinoma cells by MMAC/PTEN. Cancer Res 1999;59:2551-6. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3]