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Keywords:

  • prostate cancer;
  • emodin;
  • ascorbic acid;
  • LDL receptor-related protein 1;
  • androgen receptor;
  • anticancer;
  • antioxidant;
  • reactive oxygen species

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. Acknowledgments
  9. References

This study explores the link between the antiproliferative activity of emodin through the generation of reactive oxygen species (ROS) in various cancer cell lines and the expression of the androgen receptor (AR) in the prostate cancer cell lines LNCaP (androgen-sensitive) and PC-3 (androgen-refractory), as well as the pro-metastatic low-density lipoprotein receptor-related protein 1 (LRP1) in the above prostate cancer cells and the nonprostate cell lines A549 (lung), HCT-15 (colon) and MG-63 (bone) under normoxic and hypoxia-like conditions. Among all cell lines, emodin showed most growth inhibition in LNCaP, followed by A549. The mechanism of cytotoxicity of emodin was postulated to be the widely reported ROS generation, based on the observations of poor in vitro radical-scavenging activity and increased growth inhibition of emodin by ascorbic acid (AA) pre-treatment owing to the additive effects of ROS generation by emodin and pro-oxidant effects of AA. Emodin downregulated AR in LNCaP under normoxic and hypoxia-like conditions (simulated by CoCl2) and LRP1 under normoxia. Emodin upregulated LRP1 in other cell lines, except HCT-15, under normoxic, and even more markedly under hypoxia-like conditions. The downregulation of AR in LNCaP and upregulation of LRP1 in all cell lines, except HCT-15, under hypoxia-like conditions along with growth inhibition by emodin, suggests that emodin may be a useful therapeutic option against androgen-sensitive prostate cancer and other such LRP1-expressing cancers to attempt the targeting of the elevated LRP1 levels to allow the uptake of emodin and/or any other accompanying therapeutic agents by LRP1. Copyright © 2012 John Wiley & Sons, Ltd.


Introduction

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. Acknowledgments
  9. References

Cancer is a dreaded disease in both developed and developing countries. In India, cancer caused 6% of the total deaths in 2008 and the absolute number of deaths owing to cancer is expected to rise (Dikshit et al., 2012). Among the different types of cancer, those of the lung, prostate and colon are reported to be leading causes for concern, not only in terms of new incidences, but also in the number of deaths they cause (Jemal et al., 2011).

Prostate cancer is the most frequent cancer in males that leads to metastases to the bone and lung (Saitoh et al., 1984; Kawai et al., 2001; Jemal et al., 2011), and it has also been known to metastasize to the colon (Kabeer et al., 2007). In India, the incidence rate for prostate cancer is lower than in Western countries, but it continues to rise steadily (Yeole, 2008).

The androgen receptor (AR) plays a central role in the development of prostate cancer. Many cancer cell lines of the prostate have been found to express a mutated form of AR that enables them to interact with androgens at low concentrations or be activated by other steroidal molecules and anti-androgens. AR-dependent signaling is also implicated in hormone-refractory prostate cancer (Grossmann et al., 2001). AR has been demonstrated to be upregulated under hypoxic conditions (Park et al., 2006; Reece et al., 2012).

As solid tumors grow, their centers become hypoxic owing to the lack of blood and oxygen (Piret et al., 2002). Intratumoral hypoxia can select for cells with lowered apoptotic tendencies and induce their clonal expansion so that hypoxic tumors are found to be more malignant and metastatic (Furlan et al., 2007). Cobalt chloride (CoCl2) has been used to simulate hypoxic conditions because the signal transduction and transcriptional regulation seen in CoCl2-induced hypoxia and in vivo hypoxia are very similar (Goldberg and Schneider, 1994).

Another receptor found in prostate cancer cell lines, such as LNCaP, is LRP1 (low-density lipoprotein receptor-related protein 1), which is supposed to be upregulated in hypoxic conditions (Koong et al., 2000; Chen et al., 2007) and malignant astrocytomas, (Yamamoto et al., 1997) and promotes the in vitro invasiveness of breast cancer cells (Li et al., 1998).

This association of LRP1 with the invasiveness of cancer cells is based on its function as an endocytic receptor for proteases and protease inhibitors by which it can regulate the expression of proteins, cell signaling and the activity of other membrane receptors (Newton et al., 2005; Lindner et al., 2010). Besides being shown to promote in vitro cancer cell growth (Montel et al., 2007), LRP1 has also shown in vitro anti-apoptotic activity in neurons and Schwann cells, mediated through the PI3K-AKT pathway (Campana et al., 2006; Fuentealba et al., 2009).

Angiochem, a Canadian clinical stage biotechnology company, has reported positive results for peptide–drug conjugates that target the LRP1-mediated uptake pathway to cross the blood–brain barrier into tumor cells for the treatment of brain cancers, including glioblastoma and brain metastases, in phase 1 and 2 clinical trials (Bertrand et al., 2011; Pharmalive website, http://www.pharmalive.com/News/Index.cfm?articleid=845262, accessed 19 June 2012).

The role of emodin (6-methyl-1, 3, 8-trihydroxyanthraquinone), a molecule found in certain plants, has been explored by many researchers for its antiproliferative activity in cancer cell lines of the breast (Huang et al., 2009), colon (Lee et al., 2005), cervix (Srinivas et al., 2003), liver (Shieh et al., 2004; Hsu et al., 2010), lung (Su et al., 2005; Lai et al., 2009) and prostate (Cha et al., 2005; Zhou et al., 2006), and leukemia (Chen et al., 2002; Chun-Guang et al., 2010). It has also been reported to be chemopreventive (Koyama et al., 2002) and anti-angiogenic (Kaneshiro et al., 2006), besides enhancing the sensitivity of many cancer cell lines to chemotherapeutic agents (Dandekar and Lokeshwar, 2005; Huang et al., 2008; Yu et al., 2008; Wang et al., 2010). The underlying mechanisms of such anticancer activities of emodin include reactive oxygen species (ROS) generation (a major mechanism; Jing et al., 2002; Lai et al., 2009) and inhibition of casein kinase II (Yim et al., 1999), protein kinase C (Lee, 2001), PI3K-Cdc42/Rac1 pathway (Huang et al., 2005), ERK1/2 (Su et al., 2010) and HER-2/neu tyrosine kinase (Zhang et al., 1995).

Huang et al. (2008) showed that emodin co-treatment with As2O3, cisplatin, taxol and doxorubicin increased ROS levels and enhanced chemosensitivity in DU-145 cells compared with drug-only treatment but did not affect nontumor cells. The co-treatment was found to suppress transactivation of HIF-1α, downregulate multidrug resistance 1 to promote drug retention and, thus, facilitate cytotoxicity of emodin. ROS generation has been shown to increase with the use of CoCl2 (Huang et al., 2008).

Based on these reports, we postulated that the same LRP1 targeting strategy described by Angiochem scientists could be extended to different cancer cells, including prostate cancer cells, that express LRP1 and AR. Our hypothesis proposed the use of emodin to target LRP1 in the different cancer cells and take advantage of the ability of emodin to demonstrate cytotoxicity even in hypoxic conditions, reportedly by the suppression of transactivation of HIF-1α.

To examine the validity of such a targeting strategy for prostate and some other cancer cells, we were interested in confirming, in a preliminary in vitro study, (i) whether the anticancer potential of emodin had its basis in radical-scavenging or ROS-generating activities, (ii) given the poor in vitro radical-scavenging activity and widely reported ROS generation mechanism for emodin (Jing et al., 2002; Lai et al., 2009), the degree to which growth inhibition of the selected cell lines by emodin was influenced by pre-treatment with ascorbic acid (AA), and (iii) based on the different degrees of growth inhibition by emodin and the differences in AR and LRP1 expression in the selected cell lines, whether emodin affected the expression of these two proteins under hypoxia-like conditions induced by CoCl2 to simulate the conditions inside solid tumors of these tissues.

Materials and Methods

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. Acknowledgments
  9. References

Chemicals and Cell Culture

Agarose, AA, Dulbecco's Modified Eagle medium (DMEM) growth medium, emodin, phenylmethylsulfonyl fluoride (PMSF), resazurin sodium salt and RNAse A were purchased from Sigma-Aldrich, India. Aprotinin and leupeptin were purchased from Genetix, India. Dimethyl sulfoxide (DMSO), ethanol and methanol were purchased from SRL, India. Bromophenol blue, fetal bovine serum (FBS), penicillin–streptomycin solution (100×), proteinase K, Roswell Park Memorial Institute (RPMI)-1640 medium growth medium and trypsin–EDTA were purchased from HiMedia, India. Human cancer cell lines, A549, MG-63, HCT-15, LNCaP and PC-3, were obtained from the National Centre for Cell Sciences in Pune, India.

Antibodies and Western Blot Materials

Anti-human LRP1 antibody was a gift from Professor Gerd Birkenmeier, University of Leipzig, Germany. Anti-AR antibody (N-20) was a gift from Professor Kailash Chadha, Roswell Park Cancer Institute, USA. Mouse anti-β actin antibody (SC-47778) was purchased from Santa Cruz Biotechnology, USA. Anti-mouse and anti-rabbit alkaline phosphatase conjugated antibodies, 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) and nitro blue tetrazolium (NBT) substrate were purchased from Sigma Aldrich, India.

DPPH (2,2-Diphenyl-1-picrylhydrazile) Radical-scavenging Assay

To examine the radical-scavenging activity of emodin, the previously described DPPH radical-scavenging assay (Zubia et al., 2009; Masaldan and Iyer, 2011) was performed in a 96-well micro-plate, wherein 22 µl of various concentrations (500, 250 or 125 µg ml−1) of emodin (in methanol) were mixed with 200 µl of a freshly prepared methanolic solution of DPPH (20 mg l−1). AA was used as positive control. Scavenging was measured relative to the amount of DPPH radical formed in a mixture of 200 µl DPPH and 22 µl of methanol. The scavenging reaction was allowed to proceed in the dark for 2 h and its absorbance was read at 492 nm with a multi-well spectrophotometer (Fluostar Optima BMG Labtech GmbH). DPPH radical-scavenging activity was calculated using the formula:

  • display math

where AD is the absorbance of DPPH alone at 492 nm and At is the absorbance of the test sample or positive control (AA) at 492 nm.

Growth Inhibition Assay

MG-63 (osteosarcoma) and A549 (lung adenocarcinoma) cell lines were grown in DMEM growth medium supplemented with 10% FBS, penicillin (100 U ml−1) and streptomycin (100 µg ml−1). HCT-15 (colon carcinoma), LNCaP (androgen-sensitive prostate adenocarcinoma) and PC-3 (androgen-refractory prostate carcinoma) were grown in RPMI-1640 growth medium supplemented with 10% FBS, penicillin (100 U ml−1) and streptomycin (100 µg ml−1). Cell seeding per well for the experiment was as follows: MG-63, 8000 cells per well; A549, 2500 cells per well; LNCaP, 8000 cells per well; PC-3, 5000 cells per well and HCT-15, 4000 cells per well.

Cells were seeded in 200 µl media per well into 96-well plates and incubated at 37°C in 5% CO2 for 24 h before treatment. Emodin was dissolved in 100% DMSO to obtain a 20 mm stock solution, which was further mixed with media to obtain the desired concentrations, but maintaining the final DMSO concentration below 0.5% v/v. Doxorubicin was dissolved in PBS (50 mm) to obtain a stock concentration of 1 mm, which was dissolved in media to obtain desired concentrations for the experiments.

The growth inhibition of cells was determined, as described previously (Masaldan and Iyer, 2011), by using a resazurin-based assay. Resazurin, the active component of Alamar Blue®, is a blue redox dye that is converted into pink resorufin by oxido-reductases present in the cytoplasm, mitochondria and microsomes of viable cells. The amount of resorufin produced can be determined colorimetrically and is proportional to cell viability (Desbordes et al., 2008).

Cells were seeded in two 96-well plates (T0 and Ti plates). The T0 plate contained blank wells (media only) and untreated cells (T0). Resazurin sodium salt solution (1 mg ml−1 in 50 mm PBS) was added to all the wells in the T0 plate 24 h after seeding to achieve a final concentration of 0.1 mg ml−1 of resazurin. The T0 plate was then incubated for 4 h and read at 584 and 620 nm.

At the time of addition of resazurin to the T0 plate, doxorubicin hydrochloride (positive control, Pfizer, India) and emodin at various concentrations were added to the Ti plate. Doxorubicin hydrochloride was added in a single concentration equal to its GI50 value determined from the dose–response curves run for doxorubicin in each of the cell lines used in this study.

In addition, only medium was added to some (blank) wells without cells to account for the effects of the medium, 0.5% v/v DMSO (vehicle control), to some wells containing cells to act as a vehicle control, and some wells containing cells were left untreated (negative control). The Ti plate was then read in a similar manner as the T0 plate after 48 h of treatment of cells. The T0, Ti and control (C) values were determined using the following formulae (Boyd and Paull, 1995):

  • display math

For each treatment, the percentage of growth inhibition (%GI) was determined using the following formulae:

  • display math

Ascorbic Acid Pre-treatment Assay

AA, or vitamin C, is a known antioxidant. Its ability to scavenge ROS has been demonstrated in biochemical assays, but its role in cell-based systems is ambiguous. ROS-mediated mechanisms have been postulated for the growth inhibitory activities of emodin in cancer cell lines (Su et al., 2005; Huang et al., 2008; Lin et al., 2009). Doxorubicin, a known anticancer drug, also causes ROS generation, which contributes to its antiproliferative potential (Kotamraju et al., 2000). Hence, AA was used to determine the role played by ROS in the growth inhibition by emodin (in comparison to doxorubicin). We expected the antioxidant nature of AA to decrease growth inhibition by the two ROS generators, emodin and doxorubicin, and thus demonstrate the role played by ROS generation of emodin, in particular, in its antiproliferative activity.

To determine the effects of AA on the growth inhibitory activity of emodin or doxorubicin, the chosen panel of cell lines was subjected to 30 min pre-treatment with AA prior to 24 h treatments with emodin or doxorubicin. The cells were cultured as described previously. Cell seeding per well for this experiment was as follows: 7000 MG-63 cells per well; 3000 A549 cells per well; 10 000 LNCaP cells per well; 5000 PC-3 cells per well and 4000 HCT-15 cells per well. Cells were added in 200 µl media per well into 96 well plates and incubated at 37°C in 5% CO2 for 24 h before treatment.

Emodin was dissolved in DMSO to obtain a stock concentration of 20 mm, which was then dissolved in media to obtain the desired concentrations while ensuring that the final DMSO concentration did not exceed 0.5% v/v. AA and doxorubicin were dissolved in 50 mm PBS to obtain stock concentrations of 50 mm and 1 mm, respectively, which were dissolved in media to obtain desired concentrations for the experiments.

The growth inhibition of cells was determined as described previously. At the time of addition of resazurin to the T0 plate, wells in Ti plate were pre-treated in duplicate with AA for 30 min, after which medium was removed followed by treatments with emodin and doxorubicin hydrochloride. The AA pre-treatment concentration was 50 µm for all cell lines, except A549 for which it was 100 µm. Emodin treatments were 1.5 fold GI50 for all cell lines. Doxorubicin treatments were 2 fold GI50 for A549 and 1.5 fold GI50 for other cell lines. Wells without AA pre-treatment were also treated with the above compounds in duplicate.

Protein Extraction and Western Blotting

The effects of emodin on the expression of LRP1 in the selected cell lines were determined by western blotting. Expression of LRP1 and β-actin was determined in prostate and nonprostate cell lines and AR in only LNCaP cells (PC-3 cells did not express AR; data not shown) after treatment with cobalt chloride (CoCl2) or emodin, and treatment with both CoCl2 and emodin. Cobalt chloride is known to simulate hypoxia by stabilizing HIF-1α (Montel et al., 2007).

Cells were cultured to sub-confluent conditions and were either untreated or treated separately with emodin (50 µm) or CoCl2 (200 µm), or treated with both emodin and CoCl2 for 48 h.

Total protein from cells was isolated on ice in lysis buffer containing 50 mm PBS (pH 7.4), 1% Triton X-100, 1 mm PMSF, 20 µg ml−1 aprotinin and 20 µg ml−1 leupeptin. Protein concentration was estimated by the bichinchoninic acid assay method using a commercial kit (GeNei, India). Samples containing equal amounts of protein were subjected to SDS–PAGE using 8% polyacrylamide gel. The proteins were then transferred to nitrocellulose membrane electrophoretically at 90 V for 90 min at 4°C. The membrane was washed and blocked for 1 h in blocking buffer [5% skimmed milk in 10 mm PBS with 0.1% Tween-20 (PBST)].

After washing with PBST, the membrane was incubated with anti-human LRP1 (1:500 diluted in blocking buffer), anti-β-actin (1:500 diluted in blocking buffer) and anti-AR antibody (1:400 diluted in blocking buffer) overnight at 4°C. This was followed by washing the membrane with PBST and incubation with alkaline phosphatase-conjugated secondary antibody for 2 h at room temperature. The blot was then washed with TBST [0.5% Tween 20 in Tris-buffered saline (TBS)], developed with BCIP–NBT substrate, and then scanned (HP Image Zone). The image was analysed using the software ImageJ 1.45S (Wayne Rasband, National Institute of Health, USA).

Statistical Analysis

Statistical analysis was performed by using GraphPad Prism 5 (GraphPad Software Inc.). For the AA pre-treatment assay as well as the western analyses, one-way ANOVA, followed by post hoc Tukey's test, was used to determine statistical differences between means. All means were calculated from at least three experiments. All data are expressed as means ± SEM.

Results

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. Acknowledgments
  9. References

The Ability of Emodin to Scavenge Free Radicals as a Measure of its Antioxidant Potential

To determine whether emodin is capable of neutralizing radicals, the DPPH radical was used as a standard radical. The results of the DPPH radical scavenging assay are presented in Fig. 1, the percentage DPPH radical-scavenging activity being the percentage decrease in absorbance after treatment with AA or emodin relative to the absorbance of the DPPH radical by itself. This assay (Fig. 1) illustrates that, in comparison to AA (47% DPPH radical-scavenging activity at 25 µg ml−1), which is a potent scavenger of radicals, emodin only showed mild radical scavenging activity (6% scavenging activity at 500 µg ml−1).

image

Figure 1. DPPH radical scavenging activity of emodin and ascorbic acid. Data are represented as means ± SE (n = 3).

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Emodin Inhibits Growth in Cancer Cell Lines

To determine the effects of emodin on the growth of the selected cancer cell lines, we treated these cells for 48 h with emodin and with doxorubicin as a positive control (Fig. 2). Emodin showed a concentration-dependent antiproliferative effect on all cell lines; its GI50 was determined from the growth inhibition curves (Table 1). The growth inhibition by the anticancer compound doxorubicin is shown at various concentrations for each cell line in Fig. 2, and its GI50 values are shown in Table 1. The androgen-sensitive, AR- and LRP1-positive prostate cell line, LNCaP, was more susceptible to emodin than the androgen-refractory, AR-negative, LRP1-positive prostate, PC-3 cell line.

image

Figure 2. Growth inhibition by emodin (A) and doxorubicin (B) in A549, MG-63, HCT-15, PC-3 and LNCaP. GI50 for emodin was found to be 21.67, 50, 33.12, 36.4 and 10 µm for A549, MG-63, HCT-15, PC-3 and LNCaP, respectively. GI50 for doxorubicin was found to be 1.33, 0.35, 2, 0.813 and 0.2 µm for A549, MG-63, HCT-15, PC-3 and LNCaP respectively. Data are represented as means ± SE (n = 3).

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Table 1. GI50 for emodin and doxorubicin in prostate and nonprostate cancer cell lines
Cell lineEmodin GI50m)Doxorubicin GI50m)
Prostate  
LNCaP100.2
PC-336.40.8
Non-prostate  
A54921.71.33
HCT-1533.12
MG-6350.00.35

Among the nonprostate cell lines, A549 was found to be more sensitive than HCT-15, whereas MG-63 (the only nonprostate cell line with detectable LRP1 expression) was found to be the least sensitive to growth inhibition by emodin. Emodin was found to have lower antiproliferative potential than doxorubicin. LNCaP, among prostate cell lines, and MG-63, among the nonprostate cell lines, were most susceptible to the growth inhibitory effects of doxorubicin.

These data illustrate that emodin caused growth inhibition in different cell lines to different degrees. Even among the prostate cell lines, the AR- and LRP1-positive LNCaP was more susceptible to growth inhibition by emodin than the AR-negative, LRP1-positive PC-3. It was thus important to further explore the reasons for this difference in activity.

Ascorbic Acid Pre-treatment Experiments

Anticancer compounds are generally supposed to be either chemopreventive (radical-scavenging and, hence, preventing DNA damage and carcinogenesis) or chemotherapeutic through their effects on other cellular targets (Hickman, 1992; Galati and O'Brien, 2004). As indicated in Fig. 1, emodin was found to be a poor in vitro scavenger of radicals as compared with AA. Hence, we hypothesized that ROS generation, which has been reported to be a major mechanism of in vitro cytotoxicity of emodin in many cell lines, must also be applicable in the present scenario.

Based on the superior radical-scavenging activity (Fig. 1) and total reducing activity (data not shown) of AA, we pre-treated the different cell lines with AA and emodin or doxorubicin to examine the effects of the expected radical-scavenging activity of AA on ROS-mediated growth inhibition by emodin. The effects of AA pre-treatment on the growth inhibitory potential of doxorubicin and emodin in different cancer cell lines are presented in Fig. 3.

image

Figure 3. Effects of ascorbic acid pre-treatment on growth inhibitory activity of doxorubicin and emodin in A549, MG-63, HCT-15, PC-3 and LNCaP. Statistical analysis was carried out using ANOVA followed by post-hoc Tukey's test: extremely significant (*** P < 0.001); highly significant (** P < 0.01); and significant (* P < 0.05). Growth inhibition (GI) for each treatment was compared with the untreated control (%GI set at 0%), as shown by the asterisks directly over the bars. Also, growth inhibition was compared among the different treatments in each cell line. These pairwise comparisons are indicated by brackets with the level of significance represented as asterisks over the brackets. Data are represented as means ± SE (n = 3).

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As we studied the effects of these compounds in these AA pre-treatment experiments for 24 h instead of the 48 h period in the GI experiments, the concentrations used in these 24 h experiments were higher than the GI50 concentrations determined for these cell lines in the earlier GI experiments. Interestingly, the data show that AA by itself displayed growth inhibition in the order of LNCaP (48%) > MG-63 (31%), HCT-15 (30%) > PC-3 (21%); no growth inhibition was observed in A549.

Pairwise comparisons were performed separately for the statistical analyses of all treatments in each cell line with the respective untreated controls as well as among the various treatments. Pairwise comparisons with the untreated controls of each cell line revealed the following: (i) AA alone showed significant (P < 0.05) GI only in PC-3 cells; (ii) doxorubicin showed extremely (P < 0.001) significant GI in A549 and PC-3 cells, but not in HCT-15, MG-63 or LNCaP cells; (iii) highly (P < 0.01) or extremely significant GI was seen with emodin in all cell lines, except LNCaP; and (iv) AA pre-treatment with doxorubicin and emodin separately resulted in highly to extremely significant increase in GI in all cell lines. In Fig. 3, the significance of the GI difference between the treatment and untreated control is indicated by asterisk(s) over the bars in the respective graphs.

Pairwise comparisons among the various treatments (not relative to the untreated controls) revealed the following for each cell line:

  1. LNCaP – the only significant difference in GI was seen between AA pre-treatment with doxorubicin and doxorubicin alone.
  2. HCT-15 – AA pre-treatment with doxorubicin showed significantly higher GI than doxorubicin or AA by themselves, separately, as did AA pre-treatment with emodin in comparison to doxorubicin alone. No other comparisons showed any significant differences.
  3. MG-63 and PC-3 – AA pre-treatment with emodin showed highly to extremely significant increase in GI relative to AA and doxorubicin by themselves, and in combination. Highly significant differences were also seen between GI by emodin and GI by AA and doxorubicin separately. However, the difference in GI by doxorubicin with AA pre-treatment and by emodin alone was extremely significant only in PC-3 and not in MG-63 cells. No other comparisons showed any significant differences in GI.
  4. Except for the nonsignificant differences in GI between the doxorubicin treatments, with and without ascorbic acid pre-treatment, and the emodin treatments, with and without ascorbic acid pre-treatment, all other differences in GI were either significant or extremely significant in A549 cells.

These comparisons have been indicated by brackets with the level of significance being shown by the asterisk(s) above the brackets.

Although the expected neutralisation of the growth inhibition by emodin by AA was not observed, these data are consistent with earlier reports of AA enhancing the antiproliferative effects of antineoplastic agents (Kurbacher et al., 1996; Fromberg et al., 2011).

These results suggest that the increase in growth inhibition of some of these cell lines by emodin (and doxorubicin) owing to AA may be attributed to the additive effects of the ROS generated by emodin (and doxorubicin) and the pro-oxidant activity of AA. Also, the data demonstrate that the ROS-mediated activity of emodin is tissue-specific and that the observed in vitro growth inhibition by emodin in the selected cell lines may be influenced by the responses of different cellular factors to extracellular stimuli.

Effects of Emodin on LRP1 and AR Expression in Prostate Cancer Cells Under Normoxic and Hypoxia-like Conditions

LRP1 and AR are both proteins that are expressed by prostate cancer cells and that are upregulated in the hypoxic conditions of solid tumors (Park et al., 2006; Reece et al., 2012; Koong et al., 2000; Chen et al., 2007). Growth inhibition by emodin was greater in LNCaP cells than in PC-3 cells; AR expression was detectable only in LNCaP cells. To examine the effects of emodin on the expression of these two proteins in a setting that simulates the hypoxic conditions found inside solid tumors of the prostate, western analysis was performed on emodin-treated prostate cancer cells under normoxic and hypoxia-like conditions induced by CoCl2 (Fig. 4).

image

Figure 4. Effect of hypoxia-like conditions and emodin on LRP1 and AR expression in LNCaP and LRP1 expression in PC-3. LRP1/β-actin ratio and AR/β-actin ratio was normalized with untreated control (C) and expression was determined for CoCl2-treated (CC, 200 µm), CoCl2 (200 µm) and emodin (50 µm) cotreated (CC + E) and emodin (50 µm) treated (E) cells. Statistical analysis was carried out using ANOVA followed by post-hoc Tukey's test: extremely significant (*** P < 0.001); highly significant (** P < 0.01); and significant (* P < 0.05). Representative western blot images are shown; blotting was done at least three times. Data are represented as means ± SE.

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In AR-positive LNCaP cells (Fig. 4), AR was significantly upregulated under CoCl2-induced hypoxia-like conditions but downregulated by emodin in the presence and absence of CoCl2 when compared with the untreated control. This downregulation of AR by emodin in the presence and absence of CoCl2 was found to be extremely significant (P < 0.001) when compared with the AR levels seen in the case of CoCl2-induced hypoxia-like conditions. AR expression could not be detected in PC-3 (data not shown).

CoCl2 treatment had no significant effect by itself on LRP1 expression in LNCaP although emodin and CoCl2 co-treatment led to upregulation of LRP1 expression. Emodin treatment alone reduced LRP1 expression compared with the untreated control. The difference in LRP1 expression by emodin vs the co-treatment with CoCl2 and emodin was found to be significant.

Effects of Emodin on LRP1 Expression in Osteosarcoma, Lung and Colon Cancer Cells Under Normoxic and Hypoxia-like Conditions

The effects of emodin on the expression of LRP1 were analysed in MG-63, A549 and HCT-15 by western blotting (Fig. 5). LRP1 expression was determined along with β-actin (loading control) for untreated cells, cells under hypoxia simulated by CoCl2, and cells treated with emodin under normoxic and CoCl2-simulated hypoxic conditions.

image

Figure 5. Effects of hypoxia-like conditions and emodin on LRP1 expression in MG-63, A549 and HCT-15. LRP1/β-actin ratio was normalized with untreated control (C) and expression was determined in CoCl2-treated (CC, 200 µm), CoCl2 (200 µm) and emodin (50 µm) co-treated (CC + E) and emodin (50 µm) treated (E) cells. Statistical analysis was carried out using ANOVA followed by post-hoc Tukey's test: significant (* P < 0.05). Representative western blot images are shown; blotting was done at least three times. Data are represented as means ± SE.

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LRP1 upregulation by emodin, in the presence and absence of CoCl2, was higher in A549 cells, followed by MG-63 cells. Although a 3.5-fold increase was seen in LRP1 expression in MG-63 cells after treatment with CoCl2 alone (relative to the untreated cells), this was not statistically significant. Except in HCT-15 cells in which none of the treatments led to any change in LRP1 expression, the greatest increase in LRP1 expression was for co-treatment with emodin and CoCl2, which was significant in A549 cells.

Discussion

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. Acknowledgments
  9. References

LRP1 and AR are upregulated by hypoxia found inside growing tumors (Park et al., 2006; Reece et al., 2012; Koong et al., 2000; Chen et al., 2007). The androgen receptor plays a central role in the development of prostate cancer and LRP1 has been reported to be important for in vitro cancer cell survival as well as for the metastasis of many cancer cells. Although the overall antiproliferative activity of emodin and its effects on prostate cancer cells and AR have been extensively studied, no reports can be found on its effects on the expression of the pro-metastatic protein, LRP1.

Anticancer activity often stems from ROS-scavenging activity (chemopreventive) or ROS-mediated DNA damage to cells. Emodin has been extensively studied for its anticancer activity and several mechanisms have been reported for its growth inhibition, including ROS generation as a major mechanism. Given the antiproliferative activity of emodin, especially in hypoxic conditions, we postulated that an LRP1 targeting strategy similar to that reported by Angiochem scientists could be used in different cancer cells, such as prostate cancer cells, that expressed LRP1 and AR.

We first studied the in vitro anticancer potential of emodin in terms of radical-scavenging using the stable DPPH radical (Fig. 1). The results of the DPPH radical-scavenging assay show that, in comparison to AA, which is a potent scavenger of radicals, emodin only showed mild radical-scavenging activity. Thus, the observed antiproliferative activity of emodin in the selected cell lines may not be based, in any major way, on the radical-scavenging activity that is normally associated with chemopreventive agents but may be due to the generation of ROS which is a widely reported mechanism of action for emodin (Jing et al., 2002; Lai et al., 2009).

Next, we examined the growth inhibition of the selected cell lines by emodin with doxorubicin as a positive control (Fig. 2). Doxorubicin is widely used as an anticancer drug (Theyer et al., 1993; Litwiniec et al., 2010). One of its mechanisms of cytotoxicity is through radical generation to cause DNA damage in cancer cells. Emodin showed a concentration-dependent antiproliferative effect on all cell lines. The AR- and LRP1-positive prostate cell line, LNCaP, was more susceptible to emodin than the AR-negative, LRP-positive prostate, PC-3 cell line. Among the nonprostate cell lines, A549 was found to be more sensitive than HCT-15, whereas MG-63 was found to be the least sensitive to growth inhibition by emodin.

Based on the cell line-specific growth inhibition by emodin, poor radical-scavenging activity and the widely reported ROS generation mechanism for emodin, we examined the role of ROS generation in growth inhibition by emodin and doxorubicin (both ROS generators) by pre-treating the different cell lines with the proven radical-scavenger, AA. Concentrations of AA were selected on the basis of literature reports (Lai et al., 2009), whereas the concentrations of doxorubicin and emodin were chosen from our growth inhibition assays.

Contrary to our expectations of reduced growth inhibition by both ROS-generating molecules in the presence of the radical-scavenging AA, AA itself showed growth inhibition in all the cell lines, except A549, and increased growth inhibition by emodin and doxorubicin in all cell lines.

The growth inhibitory activity of AA is associated with its pro-oxidant potential (Rietjens et al., 2002; Chen et al., 2005) and its ability to generate hydrogen peroxide extracellularly (Fromberg et al., 2011). Our results demonstrate that the observed increase in growth inhibition by doxorubicin and emodin in cells, pre-treated with AA, was probably due to the additive effects of ROS generation by these molecules and the pro-oxidant activity of AA.

The data also demonstrate that the ROS-mediated activity of emodin is tissue-specific and that the observed in vitro growth inhibition may be influenced by the responses of different cellular factors to extracellular stimuli.

LRP1 and AR are proteins that are expressed by prostate cancer cells and that are upregulated in the hypoxic conditions of solid tumors (Park et al., 2006; Koong et al., 2000; Chen et al., 2007). Cobalt chloride (CoCl2) has been used to simulate hypoxic conditions because the signal transduction and transcriptional regulation seen in CoCl2-induced hypoxia and in vivo hypoxia are very similar (Goldberg and Schneider, 1994). ROS generation has also been shown to increase with the use of CoCl2 to mimic hypoxia (Huang et al., 2008).

As shown earlier, growth inhibition by emodin was greater in LNCaP than in PC-3 cells. In the AR-positive LNCaP cells (Fig. 4), AR was significantly upregulated under CoCl2-induced hypoxia-like conditions, decreased by emodin and CoCl2 co-treatment and further diminished by emodin treatment when compared with the untreated control. AR expression could not be detected in PC-3 (data not shown). For each of these cell lines, except for the decrease in LRP1 expression owing to emodin in LNCaP, all other treatments increased LRP1 expression relative to the untreated cells of each type.

LRP1 upregulation by emodin was higher in A549 cells and significantly higher in the presence of CoCl2, as compared with MG-63 cells. In HCT-15, none of the treatments led to a change in LRP1 expression.

Taking growth inhibition, AA pre-treatment and western analysis data together, it could be seen that the order for growth inhibition by emodin when the cells were pre-treated with AA was the same as that for LRP1 expression in hypoxia-like conditions when cells were treated with CoCl2 (calculated for each cell line relative to its own untreated cells): MG-63 > PC-3 > LNCaP > HCT-15 > A549. Except for HCT-15, LRP1 expression was upregulated the most by emodin in these cell lines in the presence of CoCl2-simulated hypoxia-like conditions, compared with untreated cells.

For the two prostate cancer cell lines, LRP1 levels after all treatments as well as the growth inhibition by emodin and doxorubicin were higher in AR-positive LNCaP cells compared with the AR-negative PC-3 cells. The order of growth inhibition by emodin for the two prostate cancer cell lines reflected the order of AR expression in the two cell lines and the increase in LRP1 in the presence of emodin and CoCl2 compared with the untreated control cells of the respective cell lines.

However, although HCT-15 cells showed no change in LRP1 expression owing to any of the treatments, it showed higher growth inhibition by emodin than MG-63 cells. A549 cells showed the greatest increase in LRP1 in the presence of emodin and CoCl2 relative to untreated A549 cells and also showed the greatest growth inhibition among the nonprostate cancer cell lines.

Thus, it would appear that upregulation of LRP1 expression, especially when accompanied by AR downregulation, by emodin in hypoxia-like conditions (as in LNCaP) is associated with good (ROS-mediated) growth inhibition by emodin. Therefore, this can be considered along with other findings to devise strategies to target LRP1 by combining emodin with other chemotherapeutic agents, either as physical mixtures or as chemical conjugates.

We expect such strategies to increase the uptake of emodin in the form of conjugates or combinations with other agents into these cancer cells (especially the AR-positive prostate cancer cells) and hence, increase the killing of cancer cells. Clearly, this concept based on this preliminary in vitro study has to be further tested both in vitro and in vivo. The results of this study thus highlight the possible involvement of AR and LRP1 in the growth inhibitory activity of emodin in the AR-positive LNCaP cells.

Conflict of Interest

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. Acknowledgments
  9. References

The authors declare no conflict of interest in this study.

Acknowledgments

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. Acknowledgments
  9. References

The authors would like to thank Professor Gerd Birkenmeier and Professor Kailash Chadha for generously providing the anti-LRP1 and anti-AR antibodies, respectively, and the Director, CBST, for facilitating this process. The authors would also like to thank Professor Anjali Karande for her valuable insights. The study was financially supported by the Department of Science and Technology, Government of India, and S.M. received financial support from the Council for Scientific and Industrial Research, Government of India through a Junior Research Fellowship (JRF) [09/844 (0002)/ 2010 EMR-I].

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  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. Acknowledgments
  9. References
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