Green Tea and Quercetin Sensitize PC-3 Xenograft Prostate Tumors To Docetaxel Chemotherapy
Green Tea and Quercetin Sensitize PC-3 Xenograft Prostate Tumors To Docetaxel Chemotherapy
Green Tea and Quercetin Sensitize PC-3 Xenograft Prostate Tumors To Docetaxel Chemotherapy
Abstract
Background: Chemotherapy with docetaxel (Doc) remains the standard treatment for metastatic and
castration-resistance prostate cancer (CRPC). However, the clinical success of Doc is limited by its chemoresistance
and side effects. This study investigated whether natural products green tea (GT) and quercetin (Q) enhance
the therapeutic efficacy of Doc in CRPC in mouse models.
Methods: Male severe combined immunodeficiency (SCID) mice (n = 10 per group) were inoculated with
androgen-independent prostate cancer PC-3 cells subcutaneously. When tumors were established the intervention
started. Mice were administered with GT + Q, Doc 5 mg/kg (LD), GT + Q + LD Doc, Doc 10 mg/kg (HD) or control.
The concentration of GT polyphenols in brewed tea administered as drinking water was 0.07 % and Q was
supplemented in diet at 0.4 %. Doc was intravenously injected weekly for 4 weeks, GT and Q given throughout
the study.
Results: GT + Q or LD Doc slightly inhibited tumor growth compared to control. However, the combination of
GT and Q with LD Doc significantly enhanced the potency of Doc 2-fold and reduced tumor growth by 62 %
compared to LD Doc in 7-weeks intervention. A decrease of Ki67 and increase of cleaved caspase 7 were observed
in tumors by the mixture, along with lowered blood concentrations of growth factors like VEGF and EGF. The
mixture significantly elevated the levels of tumor suppressor mir15a and mir330 in tumor tissues. An increased
risk of liver toxicity was only observed with HD Doc treatment.
Conclusions: These results provide a promising regimen to enhance the therapeutic effect of Doc in a less toxic
manner.
Keywords: Green tea polyphenol, Quercetin, Docetaxel, Prostate cancer, Combination
© 2016 Wang et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Wang et al. Journal of Experimental & Clinical Cancer Research (2016) 35:73 Page 2 of 11
Green tea (GT) is produced from the leaves of the plant for 5 min. The GT water contained 0.07 % of the major
Camellia sinensis. The anti-cancer activities of GT have GTPs including (mg/L): EGC 204 ± 4, EGCG 388 ± 12, EC
been demonstrated in several cancers including prostate, 44 ± 2, ECG 64 ± 7, and catechin 7 ± 1. GT was freshly
mammary gland, colon, pancreas, liver, esophagus and prepared three times per week on Monday, Wednesday
liver cancer [6, 7]. The major bioactive components of GT and Friday and administered as drinking water ad libitum.
are GT polyphenols (GTPs), mainly including (-)-epigallo- Q (Sigma-Aldrich, St Louis, MO) was supplemented in
catechin (EGC), (-)-epigallocatechin-3-gallate (EGCG), AIN-93G diet at a concentration of 0.4 % by Dyets Inc.
(-)-epicatechin (EC), and (-)-epicatechin-3-gallate (ECG), (Bethlehem, PA). Docetaxel Injection (Besse Medical,
with EGCG as the most abundant and most bioactive West Chester, OH) was diluted in sodium chloride for tail
component [6]. Quercetin (Q) is a flavonoid widely found vein injection.
in vegetables and fruits particularly in onions, apples, and
red wine. We were able to demonstrate in vitro that the Animal study
combination of GT and Q with Doc synergistically All procedures carried out in mice were approved by the
enhanced the anti-proliferative effect in androgen- Charles R. Drew University of Medicine and Science
independent PC-3 and LAPC-4-AI cells [8]. The com- Institutional Animal Care and Use Committee. Male
bined effect was associated with increased apoptosis and severe combined immunodeficiency (SCID) mice (Charles
cell cycle arrest in both cell lines [8]. Apoptosis through River Laboratories) of 5–7 weeks old were acclimated to a
the mitochondrial (intrinsic) pathway can be initiated by sterilized AIN-93G diet (Dyets Inc.) and water for seven
chemotherapy or other stimuli such as reduced cytokines/ days. Mice were inoculated subcutaneously with 5x105
growth factors. The pro-apoptotic BCL2 family proteins androgen-independent PC-3 human prostate cancer cells.
like Bax, Bak and Bcl-2-associated death promotor (Bad) The PC-3 cell line (ATCC® CRL-1435™) was purchased
are important mediators of these signals. Dephosphory- from American Type Culture Collection (Manassas, VA),
lated Bad forms a heterodimer with the anti-apoptotic where the cells were authenticated by STR DNA profiling
BCL2 family members Bcl-2 and Bcl-xL, which allows Bax and tested as mycoplasma free. PC-3 cells were main-
and Bak to aggregate and initiate apoptosis [8]. The subse- tained in RPMI 1640 medium supplemented with 10 %
quent activation of caspases including caspase 3 and 7 (v:v) of fetal bovine serum, 100 IU/mL of penicillin and
leads to the cleavage of their substrate poly (ADP-ribose) 100 μg/mL of streptomycin at 37 °C in a 5 % CO2 incuba-
polymerase 1 (PARP1), which is a hallmark of apoptosis tor, and were tested periodically for mycoplasma contam-
[9]. Apoptosis through mitochondria can be inhibited by ination. The passage of the PC-3 cell line used for the
survival signals, such as growth factors and cytokines, inoculation was <10. Four weeks after inoculation when
through activation of anti-apoptotic pathways such as the tumors reached a volume of 50–80 mm3 the intervention
NFκB pathway. In cytoplasm, NFκB is bound and inhib- treatment started. Mice were randomly assigned to one of
ited by the inhibitor of NFκB protein (IκB). Once IκB is the five groups (n = 10 per group) including: 1) control, re-
phosphorylated, NFκB will be released and translocated to ceiving AIN-93G diet + water + vehicle (sodium chloride);
nucleus where it induces the expression of target genes to 2) GT + Q, receiving Q diet + GT + vehicle; 3) low dose
promote cell proliferation and survival [10]. (LD) Doc, receiving AIN-93G diet + water + 5 mg/kg Doc;
The multiple-targeting activities of GT and Q in anti- 4) GT + Q + LD Doc, receiving Q diet + GT + 5 mg/kg
carcinogenesis, particularly by targeting the NFκB and Doc ; and 5) high dose (HD) Doc, receiving AIN-93G
pro-apoptotic pathways, make them ideal candidates to be diet + water + 10 mg/kg Doc [11]. Doc was administered
combined with Doc to enhance the therapeutic effect in a once a week for 4 weeks, GT and Q throughout the study.
less-toxic manner. In in vitro studies we have observed a Tumor size was measured once a week with calipers.
synergistic anti-proliferative effect by combining GT and Tumor volume was calculated using the formula: length x
Q with Doc [8]. The present study was performed to width x height x 0.5236 [12]. Mouse body weight was
confirm the combined effect of the mixture in vivo in a measured once a week. Mice in the control and GT + Q
xenograft mouse model. This study provides evidence that groups were sacrificed when they had received 5-weeks
the combination of GT, Q and Doc enhances the chemo- intervention treatment in observance of the institutional
therapeutic activity of Doc and encourages future use in guideline on tumor size. Other groups were sacrificed
clinical practice in treatment of CRPC with enhanced drug 2 weeks later.
efficacy and reduced side effects.
Apoptosis signaling antibody array assay
Methods Eight tumor samples were randomly selected from each
Preparation of GT water, Q diet and Doc solution group for all the following molecular analyses in tumors.
GT was prepared by brewing one tea bag (Celestial A slide-based antibody array was used for simultaneous
Seasonings, Boulder, CO) in 240 mL of boiling water detection of 19 important signaling molecules involved
Wang et al. Journal of Experimental & Clinical Cancer Research (2016) 35:73 Page 3 of 11
in stress response and apoptosis, using a PathScan Stress with goat serum the slides were incubated with
and Apoptosis Signaling Antibody Array kit (Cell Signal- monoclonal Ki67 antibody (DAKO North America Inc.,
ing Technology, Danvers, MA) following the manufac- Carpineteria, CA). The slides were counterstained with
turer’s instructions. Each of these molecules was detected hematoxylin. Slides were digitized on a ScanScope AT
in duplicate on the same array arranged as shown in (Aperio Technologies, Inc., Vista, CA) and morphometric
Fig. 2c, and the identity of each numbered molecule listed analysis performed with Definiens’ Tissue Studio (Defini-
in Fig. 2d. Briefly, total protein was extracted from tumor ens Inc., Parsippany, NJ) to determine the percentage of
tissues using RIPA buffer (Santa Cruz Technology, CA), Ki67 positive cells in a non-biased method.
and diluted to 0.5 mg/mL in Array Diluent Buffer pro-
vided by the kit. The protein samples were incubated with Measurement of growth factors in blood
the array antibodies overnight. A Detection Antibody Several growth factors play important roles in tumor cell
Cocktail was added to the samples, followed by the proliferation and angiogenesis. The blood levels of growth
addition of HRP-linked Streptavidin and substrate. Protein factors were measured using a Human Growth Factor
was visualized using a ChemiDoc XRS chemiluminescence ELISA Strip II kit (Signosis, inc., Santa Clara, CA). Eight
detection and imaging system (Bio-Rad Laboratories, blood samples were randomly selected from each group
Irvine, CA). The density of the spots were quantitated for this analysis. The kit allows a simultaneous quan-
using Quantity One software (Bio-Rad Laboratories) and tification of 8 growth factors, including VEGF, epider-
normalized to the α-tubulin levels. Each sample was done mal growth factor (EGF), platelet-derived growth factor
in duplicate. (PDGF)-BB, nerve growth factor (NGF)-β, stem cell factor
(SCF), tumor necrosis factor (TNF)-α, fibroblast growth
Western blot analysis of protein markers of apoptosis and factor (FGF)-β, and transforming growth factor (TGF)-β.
angiogenesis Briefly, serum samples were diluted by 1:10 and 100 μl
Total protein was extracted from tumor tissues using was added to each well of the 8-wells strip coated with dif-
RIPA lysis buffer (Santa Cruz, CA). The Western blot ferent antibodies. After 1 h incubation the wells were aspi-
procedure was described previously [13]. Briefly, 50 μg rated and washed. A biotin-labeled antibody was added to
of protein was separated on a 4–12 % Bis-Tris gel the samples followed by streptavidin-HRP conjugate. The
(Invitrogen, Carlsbad, CA), electrotransferred to nitrocel- luminescence was measured after adding a HRP substrate
lulose membranes and blocked in Tris-buffered saline solution. The expression level of these growth factors is
with 0.1 % Tween 20 and 5 % nonfat milk for 1 h at room proportional to the luminescent intensity.
temperature. Membranes were incubated with primary
antibodies for the detection of Bax (sc-493), Bcl-2 qRT-PCR analysis of microRNA expression
(sc-509), and vascular endothelial growth factor (VEGF, Total microRNA (miRNA) was extracted from tumor
sc-152), respectively. GAPDH protein was used as tissues using a miRNeasy mini kit (Qiagen), and re-
loading control. Images were visualized and quantified versely transcribed to cDNA using miScript II RT kit
using a ChemiDoc XRS chemiluminescence detection (Qiagen). Specific primers were provided by miScript
system (Bio-Rad Laboratories). Each sample was done Primer Assays for miR-15a (MS00003178), miR-330
in duplicate. (MS00009450), and miR-19b (MS00031584). The quan-
titative real-time (qRT)-PCR procedure was described
Tissue microarray and immunohistochemical analysis of previously [15]. Briefly, a miScript SYBR Green PCR kit
proliferation (Qiagen) was used for the reaction. The 25 μL final vol-
A section of each tumor was fixed in 10 % phosphate ume of reaction contained 12.5 μL of 2x QuantiTect
buffered formalin and paraffin-embedded for tissue SYBR Green PCR Master Mix, 2.5 μL of 10x miScript
microarray and immunohistochemical detection. The Universal Primer, 2.5 μL of 10x miScript Primer Assay,
procedures for tissue array assembling and immunohis- 2.5 μL of template cDNA, and RNase-free water. The
tochemical detection were described before [14]. Briefly, reaction mixture was incubated at 95 °C for 15 min,
a total of 6 cylindrical cores 1.0 mm in diameter were followed by 40 cycles of 94 °C for 15 s, 55 °C for 30 s,
transferred from each donor block to two new paraffin and 70 °C for 30 s. Mature miRNA expression was
array blocks. Four-micron sections were cut from each calculated using the 2-ΔΔCt method in normalization to
array for staining. The slides were prepared for immuno- human RNU6-2 snRNA (MS00033740). Each sample was
histochemistry by deparaffinizing and dehydrating, done in triplicate.
followed by an alcohol series and washing in PBS. All
samples were incubated in 3 % H2O2 to eliminate en- Blood ALT and AST measurement
dogenous peroxidase activity, and antigens were retrieved Side effect is one of the major issues that limit the dosage
by boiling the slides in a microwave oven. After blocking and efficacy of most chemotherapy drugs. The damage on
Wang et al. Journal of Experimental & Clinical Cancer Research (2016) 35:73 Page 4 of 11
liver is frequently found with Doc treatment and is an using assay kits (Sigma-Aldrich). 20 ul of serum was used
indicator of Doc toxicity [16]. The blood levels of liver for each reaction and each sample was done in triplicate.
enzymes like alanine transaminase (ALT) and aspartate
transaminase (AST) are commonly used as markers of Liver pathological examination
liver damage. The possibility of treatment as well as A section of liver tissue was fixed in 10 % phosphate
cancer related liver damage was evaluated by both blood buffered formalin and paraffin-embedded. Eight liver
enzyme and liver pathological evaluations in the present samples were randomly selected from each group for
study. Blood samples were collected at mouse sacrificing pathological examination. Slides were cut and stained
and serum separated. Eight blood samples were randomly with H&E. The pathologist Dr. Elshimali did the liver
selected from each group for this analysis. The blood ALT pathological examination in a blind manner. The liver
and AST levels were determined by measuring their col- condition was graded based on the degree of inflamma-
orimetric products pyruvate and glutamate, respectively, tion, piecemeal or bridging necrosis with the following
Fig. 1 GT and Q in combination with Doc enhanced the inhibition of PC-3 xenograft tumor growth in SCID mice. Male SCID mice were
inoculated subcutaneously with 5x105 androgen-independent PC-3 prostate tumor cells. The intervention treatments started 4 weeks later when
tumors were established. Doc was injected through tail vein once a week for 4 weeks. GT and Q were administered throughout the study. Tumor
size was measured using caliper and volume calculated using the formula: length x width x height x 0.5236. Data are presented as mean ± SE for
tumor volume (a) or mean ± SD for tumor weight (b). N = 10 per group. GT, green tea; Q, quercetin; Doc 5, docetaxel at 5 mg/kg iv; Doc 10,
docetaxel at 10 mg/kg iv. Different letters at each time points represent significant difference between treatments (P < 0.05)
Wang et al. Journal of Experimental & Clinical Cancer Research (2016) 35:73 Page 5 of 11
criteria: Grade 0: no/minimal inflammation; Grade 1: por- were considered significant if P < 0.05. SPSS (Version 20,
tal inflammation or lobular inflammation without necro- Chicago, IL) was used for all statistical analysis.
sis; Grade 2: mild periportal inflammation and piecemeal
necrosis or focal hepatocellular necrosis; Grade 3: moder- Results
ate periportal inflammation and piecemeal necrosis or se- Enhanced inhibition of PC-3 xenograft tumor growth
vere focal cell damage; Grade 4: severe periportal Different profiles of tumor growth were observed between
inflammation and piecemeal necrosis or bridging necrosis. treatments. Only treatments with the HD Doc and
combination of GT + Q + LD Doc significantly decreased
Statistical analysis tumor growth throughout the intervention (Fig. 1a). LD
Data were expressed as mean ± standard deviation (SD) Doc treatment initially induced a significant inhibition of
or mean ± standard error (SE). Comparison of means tumor growth but after week 8 tumor growth accelerated
was performed by one-way analysis of variance (ANOVA) (Fig. 1a). GT + Q treatment did not inhibit tumor growth
with Tukey’s posttest for paired comparison. Differences significantly compared to control (Fig. 1a). In summary at
Fig. 2 Modulation of apoptosis related signaling proteins using antibody array assay. Eight tumor samples were randomly selected from each
group for this analysis. Total protein was extracted from the tumor tissues. A slide-based antibody array was used for simultaneous detection of
19 signaling molecules (c and d) involved in stress response and apoptosis using a PathScan Stress and Apoptosis Signaling Array kit. Each protein
was arranged in duplicate. The names of the proteins with significant changes in concentration were indicated on the images (a) and their concentrations
quantified (b). Data are presented as mean ± SD. GT, green tea; Q, quercetin; Doc 5, docetaxel at 5 mg/kg iv. Columns with different letters represent
significant difference between treatments (P < 0.05)
Wang et al. Journal of Experimental & Clinical Cancer Research (2016) 35:73 Page 6 of 11
week 9, the tumor growth was inhibited by 26 % (GT + significantly effective to modulate these pathways in the
Q), 52 % (LD Doc), and 85 % (GT + Q + LD Doc) tumor tissues compared to control (Fig. 2a and b). Both
compared to control. At week 11, the mixture of all three LD Doc alone and its combination with GT and Q signifi-
chemicals reduced the tumor growth by 62 % compared cantly decreased the phosphorylation of Bad protein com-
to LD Doc alone. The combined effect of the mixture was pared to control. However, a significant increase of
comparable to that by a two-fold higher dose of Doc cleaved PARP (by 30 % compared to control) and cleaved
(HD Doc) (Fig. 1a). A consistent pattern was observed caspase 7 (by 52 %) and decrease of IκBα (by 32 %) were
between tumor weight and tumor volume (Fig. 1b). only achieved by the combination of all three chemicals
(Fig. 2a and b). Western blot analysis of Bax and Bcl-2
Increased tumor cell apoptosis protein expression in tumor tissues found no significant
A protein array analysis was performed to determine the difference in Bax/Bcl-2 ratio with the treatment of GT + Q
effect of different treatments on apoptotic signaling path- or LD Doc alone compared to control. However, the com-
ways, including molecules involved in the mitochondrial bination of GT, Q and LD Doc significantly increased
(intrinsic) pathway such as Bad, caspases, and PARP, and the ratio of Bax/Bcl-2 compared to other treatments
NFκB pathway. The treatment with GT + Q was not (Fig. 3a and b).
Fig. 3 Increased Bax/Bcl-2 ratio and decreased Ki67 protein expression by GT, Q and Doc in combination. Eight tumor samples were randomly
selected from each group for this analysis. The protein expression of Bax and Bcl-2 in tumor tissues was detected by Western blot. Three representative
protein images from each group were demonstrated (a). The ratios of Bax to Bcl-2 are presented as mean ± SD (b). A section of tumor tissue was
formalin-fixed and paraffin-embedded for tissue microarray and immunohistochemical detection. Slides were cut from the arrays and incubated with
primary antibodies for detection of Ki67 (c). The slides were counterstained with hematoxylin. Nuclei were stained in blue and Ki67 in brown color.
The positive rates of Ki67 nuclear staining are presented as mean ± SD (d). GT, green tea; Q, quercetin; Doc 5, docetaxel at 5 mg/kg iv. Columns with
different letters represent significant difference between treatments (P < 0.05)
Wang et al. Journal of Experimental & Clinical Cancer Research (2016) 35:73 Page 7 of 11
Decreased tumor cell proliferation SCF, TNF-α, FGF-β, and TGF-β, have been demon-
Tumor cell proliferation under different treatments was strated in many studies [17–19]. Several of them such as
evaluated by the levels of the proliferation marker nu- EGF and TGF-β were found to be involved in the che-
clear Ki67 protein in tumor tissues using tissue micro- moresistance to Doc [20, 21]. The capacity of GT, Q and
array and immunohistochemical analysis. There was no Doc in reducing the expression of these growth factors
significant changes in tumor Ki67 levels in GT + Q or were analyzed in eight tumor tissues from each group.
LD Doc alone groups compared to control. However, There was a slight but nonsignificant decrease in the
the combination of GT and Q with LD Doc significantly blood levels of these growth factors with GT + Q or LD
decreased the Ki67 levels compared to both control and Doc treatment (Fig. 4a). However, the combination of
LD Doc (Fig. 3c and d). GT, Q and LD Doc significantly decreased the levels of
most growth factors including VEGF, EGF, NGF-β, SCF,
Downregulation of tumor growth factors TNF-α, FGF-β, and TGF-β, by 40 to 70 % compared to
The tumor stimulating activities of the eight analyzed control, with a nonsignificant reduction of PDGF-BB
growth factors, including VEGF, EGF, PDGF-BB, NGF-β, level (Fig. 4a). To confirm the correlation between the
Fig. 4 Decreased blood concentrations of tumor growth factors and tumor concentrations of VEGF expression by combining GT and Q with Doc. Blood
samples were collected from each mouse at sacrificing and serum separated. Eight blood samples were randomly selected from each group for this
analysis. The levels of 8 growth factors in blood were simultaneously measured using a Human Growth Factor ELISA Strip II kit. Data are presented as
mean ± SD (a). Eight tumor samples were randomly selected from each group for Western blot analysis of VEGF protein expression. Three representative
protein images from each group were demonstrated (b). The relative concentrations of VEGF in tumor tissues are presented as mean ± SD (c). GT, green
tea; Q, quercetin; Doc 5, docetaxel at 5 mg/kg iv. Columns with different letters represent significant difference between treatments (P < 0.05)
Wang et al. Journal of Experimental & Clinical Cancer Research (2016) 35:73 Page 8 of 11
blood and tissue concentrations of these growth factors, compared to control, however, the level of miR-19b
the expression of VEGF in tumor tissues was analyzed was reduced to the control level when combining LD
by Western blot. The pattern of VEGF expression in Doc with GT and Q (Fig. 5).
tumor tissues was consistent with that in blood under
different treatments (Fig. 4b and c). Reduced risk of liver damage by the combination treatment
Liver toxicity is one of the common and dose-limiting
Modulation of miRNA expression side effects with Doc treatment. The evaluation of liver
Several phytochemicals including EGCG and Q have toxicity in this study was performed by measuring blood
demonstrated the ability to modulate the expression of levels of the liver enzymes ALT and AST as well as by a
miRNA, a class of small non-coding RNAs that interact pathological evaluation of liver tissues. There were no
with target mRNA to negatively regulate the gene ex- significant changes in blood ALT level with GT + Q, LD
pression at the post-transcriptional level [22–24]. The Doc, or their combination treatment compared to con-
importance of miRNAs in carcinogenesis has been dem- trol, as indicated by the formation of pyruvate (Fig. 6a).
onstrated by many studies. MiRNAs have been found to However, a significantly elevated level of blood ALT was
be involved in tumor growth, invasion, angiogenesis, and observed with the HD Doc treatment. The blood AST
immune evasion, and they are potential therapeutic levels were not significantly different among groups
targets [25]. To investigate whether miRNAs are respon- (data not shown). The liver tissues were pathologically
sive to the combination treatment of GT, Q and Doc, we examined (Fig. 6b) and graded as following: 2.2 ± 0.5
selected three candidate miRNAs that have been shown (control), 2.2 ± 0.4 (GT + Q), 2.6 ± 0.8 (LD Doc), 2.7 ± 0.6
to be involved in prostate cancer, including two tumor (GT + Q + LD Doc), and 3.2 ± 0.9 (HD Doc), with a sig-
suppressor miR-15a and miR-330 and an oncomiR miR- nificantly higher grade in HD Doc group compared to
19b. A different profile of miRNA expression was observed control or GT + Q group.
in tumor tissues with GT + Q, Doc or their combination
treatment. Both GT + Q and LD Doc alone demonstrated Discussion
a non-significant trend to increase the expression of This study demonstrates that a combination of GT and
miR-15a compared to control, while their combin- Q with Doc significantly enhanced the potency of Doc
ation significantly elevated the level of miR-15a (Fig. 5). by 2-fold in inhibition of PC-3 xenograft prostate tumor
Similarly, the mixture of all three chemicals significantly growth in SCID mice. The combined effect of the mix-
increased miR-330 expression compared to other treat- ture was associated with increased apoptosis, decreased
ments. A significantly increased expression of the proliferation, and enhanced modulation of multiple sig-
oncogenic miR-19b was observed with LD Doc alone naling pathways. Tumors may have hundreds of gene
mutations and dysfunctions and alternate signaling path-
ways may compensate for lack of function, thus a therapy
targeting single or few pathways may not be effective and
chemoresistance may develop soon. This is one of the
major causes of the failure of most chemotherapy drugs
including Doc [26]. Effort has been made to test the com-
binations of several other chemotherapy drugs such as
vinorelbine and capecitabine with Doc [27]. However, no
superiority to Doc/prednisone effect has been shown in
phase III trials. In addition, these drug-drug combinations
increase the challenge of adverse effects [27].
Natural compounds like GTPs and Q target multiple
signaling pathways and events in anti-carcinogenesis,
therefore in combination with chemotherapy drugs nat-
ural compounds may be able to provide a systemic con-
Fig. 5 Modulation of miRNA expression. Eight tumor samples were trol of the disease with enhanced therapeutic effect at
randomly selected from each group for this analysis. Total miRNA
lower doses of the drug. Previous studies showed that
was extracted from tumor tissues, reversely transcribed to cDNA and
quantified using quantitative real-time PCR. Mature miRNA expression oral administration of GTPs in drinking water equivalent
was calculated using the 2-ΔΔCt method in normalization to human to a realistic dose for human consumption (4–6 cups of
RNU6-2 snRNA. Each sample was done in triplicate. Data are presented tea daily for an average adult human) significantly inhib-
as mean ± SD. GT, green tea; Q, quercetin; Doc 5, docetaxel at 5 mg/kg ited the growth of xenograft prostate tumors in mouse
iv. Columns with different letters represent significant difference
models and progression of prostate tumors in transgenic
between treatments (P < 0.05)
mice as demonstrated by our group and other
Wang et al. Journal of Experimental & Clinical Cancer Research (2016) 35:73 Page 9 of 11
Fig. 6 GT and Q reduced the risk of liver damage in enhancing the therapeutic effect of Doc. The liver condition was evaluated with both
blood level of liver enzymes and liver pathological examination. Eight blood and liver samples were randomly selected from each group for this
analysis. The blood alanine transaminase (ALT) level was determined by measuring its colorimetric product pyruvate using assay kit (a). Data
are presented as mean ± SD. For the liver pathological examination, a section of liver tissue was fixed in 10 % phosphate buffered formalin and
paraffin-embedded. Slides were cut and stained with H&E. The liver condition was graded based on the degree of inflammation, piecemeal or
bridging necrosis, and representative images from each group were presented (b)
investigators [10, 11]. Evidence from human studies is in- including the induction of apoptosis and the inhibition
consistent, however, several recent clinical trials have of proliferation and insulin-like growth factor (IGF)-1
demonstrated encouraging results [12, 13]. In a phase II pathway [19–22].
clinical trial of tea in prostate cancer patients, we found The doses of GT and Q used in this study are physiolo-
that the consumption of GT at 6 cups a day for 1 month gically relevant and safe for human consumption [28, 29].
significantly decreased serum prostate-specific antigen The GT dose used is equivalent to the consumption of
(PSA) levels and nuclear NFκB staining in radical prosta- 5–6 cups of GT per day for an adult human, and Q
tectomy tissue as compared to control [14]. However, an 2 g per day for an adult. The same combination of
analysis of GTPs in the prostate tissues revealed that GT and Q was able to significantly enhance the inhib-
about 50 % of EGCG was methylated into less active me- ition of tumor growth compared to GT or Q alone in
tabolites, which limits the anti-cancer activity of GT [15]. SCID mice implanted with androgen-dependent LAPC-4
We found that the combined use of Q, a natural inhibitor prostate cancer cells [14]. However, the administration of
of both catechol-O-methyltransferase (COMT) and multi- GT + Q without Doc was not significantly effective in
drug resistance-associated proteins (MRPs), with GT sig- inhibition of PC-3 xenograft tumor growth in the present
nificantly increased the bioavailability and cellular uptake study. These results are consistent with our previous in
of GTPs and decreased their methylation in vitro and vitro observations that PC-3 cells were less sensitive to
in vivo, leading to a synergistically enhanced inhib- GT and Q compared to androgen-dependent LNCaP
ition of xenograft prostate tumor growth in SCID mice and LAPC-4 cells [30]. Treatment with LD Doc was
[16–18]. Q itself has exhibited chemopreventive activities initially effective, while tumors started to grow rapidly
especially in prostate cancer through multiple mechanisms, after the last dose of Doc and eventually tumors were
Wang et al. Journal of Experimental & Clinical Cancer Research (2016) 35:73 Page 10 of 11
8. Wang P, Henning SM, Heber D, Vadgama JV. Sensitization to docetaxel 32. Li J, Xiang S, Zhang Q, Wu J, Tang Q, Zhou J, Yang L, Chen Z, Hann SS.
in prostate cancer cells by green tea and quercetin. J Nutr Biochem. Combination of curcumin and bicalutamide enhanced the growth
2015;26:408–15. inhibition of androgen-independent prostate cancer cells through SAPK/JNK
9. Boucher D, Blais V, Denault JB. Caspase-7 uses an exosite to promote and MEK/ERK1/2-mediated targeting NF-kappaB/p65 and MUC1-C. J Exp
poly(ADP ribose) polymerase 1 proteolysis. Proc Natl Acad Sci U S A. Clin Cancer Res. 2015;34:46.
2012;109:5669–74. 33. Singh BN, Shankar S, Srivastava RK. Green tea catechin, epigallocatechin-3-
10. Hoesel B, Schmid JA. The complexity of NF-kappaB signaling in gallate (EGCG): mechanisms, perspectives and clinical applications.
inflammation and cancer. Mol Cancer. 2013;12:86. Biochem Pharmacol. 2011;82:1807–21.
11. Fizazi K, Sikes CR, Kim J, Yang J, Martinez LA, Olive MC, Logothetis CJ, 34. Bonci D, Coppola V, Musumeci M, Addario A, Giuffrida R, Memeo L,
Navone NM. High efficacy of docetaxel with and without androgen D’Urso L, Pagliuca A, Biffoni M, Labbaye C, et al. The miR-15a-miR-16-1
deprivation and estramustine in preclinical models of advanced prostate cluster controls prostate cancer by targeting multiple oncogenic activities.
cancer. Anticancer Res. 2004;24:2897–903. Nat Med. 2008;14:1271–7.
12. Seeram NP, Aronson WJ, Zhang Y, Henning SM, Moro A, Lee RP, Sartippour M, 35. Lee KH, Chen YL, Yeh SD, Hsiao M, Lin JT, Goan YG, Lu PJ. MicroRNA-330
Harris DM, Rettig M, Suchard MA, et al. Pomegranate ellagitannin-derived acts as tumor suppressor and induces apoptosis of prostate cancer cells
metabolites inhibit prostate cancer growth and localize to the mouse prostate through E2F1-mediated suppression of Akt phosphorylation. Oncogene.
gland. J Agric Food Chem. 2007;55:7732–7. 2009;28:3360–70.
13. Wang P, Heber D, Henning SM. Quercetin increased bioavailability and 36. Mao Y, Chen H, Lin Y, Xu X, Hu Z, Zhu Y, Wu J, Xu X, Zheng X, Xie L.
decreased methylation of green tea polyphenols in vitro and in vivo. microRNA-330 inhibits cell motility by downregulating Sp1 in prostate
Food Funct. 2012;3:635–42. cancer cells. Oncol Rep. 2013;30:327–33.
14. Wang P, Vadgama JV, Said JW, Magyar CE, Doan N, Heber D, Henning SM. 37. Tian L, Fang YX, Xue JL, Chen JZ. Four microRNAs promote prostate cell
Enhanced inhibition of prostate cancer xenograft tumor growth by proliferation with regulation of PTEN and its downstream signals in vitro.
combining quercetin and green tea. J Nutr Biochem. 2014;25:73–80. PLoS One. 2013;8:e75885.
15. Wang P, Phan T, Gordon D, Chung S, Henning SM, Vadgama JV. Arctigenin
in combination with quercetin synergistically enhances the antiproliferative
effect in prostate cancer cells. Mol Nutr Food Res. 2015;59:250–61.
16. King PD, Perry MC. Hepatotoxicity of chemotherapy. Oncologist.
2001;6:162–76.
17. Cheng Y, Guo Y, Zhang Y, You K, Li Z, Geng L. MicroRNA-106b is involved in
transforming growth factor beta1-induced cell migration by targeting
disabled homolog 2 in cervical carcinoma. J Exp Clin Cancer Res. 2016;35:11.
18. Yang T, Shi R, Chang L, Tang K, Chen K, Yu G, Tian Y, Guo Y, He W, Song X,
et al. Huachansu suppresses human bladder cancer cell growth through the
Fas/Fasl and TNF- alpha/TNFR1 pathway in vitro and in vivo. J Exp Clin
Cancer Res. 2015;34:21.
19. Witsch E, Sela M, Yarden Y. Roles for growth factors in cancer progression.
Physiology (Bethesda). 2010;25:85–101.
20. Hour TC, Chung SD, Kang WY, Lin YC, Chuang SJ, Huang AM, Wu WJ,
Huang SP, Huang CY, Pu YS. EGFR mediates docetaxel resistance in human
castration-resistant prostate cancer through the Akt-dependent expression
of ABCB1 (MDR1). Arch Toxicol. 2015;89:591–605.
21. Magadoux L, Isambert N, Plenchette S, Jeannin JF, Laurens V. Emerging
targets to monitor and overcome docetaxel resistance in castration resistant
prostate cancer (review). Int J Oncol. 2014;45:919–28.
22. Milenkovic D, Jude B, Morand C. miRNA as molecular target of polyphenols
underlying their biological effects. Free Radic Biol Med. 2013;64:40–51.
23. Boesch-Saadatmandi C, Wagner AE, Wolffram S, Rimbach G. Effect of
quercetin on inflammatory gene expression in mice liver in vivo - role of
redox factor 1, miRNA-122 and miRNA-125b. Pharmacol Res. 2012;65:523–30.
24. Milenkovic D, Deval C, Gouranton E, Landrier JF, Scalbert A, Morand C,
Mazur A. Modulation of miRNA expression by dietary polyphenols in apoE
deficient mice: a new mechanism of the action of polyphenols. PLoS One.
2012;7:e29837.
25. Hayes J, Peruzzi PP, Lawler S. MicroRNAs in cancer: biomarkers, functions
and therapy. Trends Mol Med. 2014;20:460–9.
26. Hasima N, Aggarwal BB. Cancer-linked targets modulated by curcumin.
Int J Biochem Mol Biol. 2012;3:328–51.
27. Vishnu P, Tan WW. Update on options for treatment of metastatic
castration-resistant prostate cancer. Onco Targets Ther. 2010;3:39–51. Submit your next manuscript to BioMed Central
28. Henning SM, Wang P, Said JW, Huang M, Grogan T, Elashoff D, Carpenter and we will help you at every step:
CL, Heber D, Aronson WJ. Randomized clinical trial of brewed green and
black tea in men with prostate cancer prior to prostatectomy. Prostate. • We accept pre-submission inquiries
2015;75:550–9. • Our selector tool helps you to find the most relevant journal
29. Shoskes DA, Zeitlin SI, Shahed A, Rajfer J. Quercetin in men with category III
• We provide round the clock customer support
chronic prostatitis: a preliminary prospective, double-blind, placebo-
controlled trial. Urology. 1999;54:960–3. • Convenient online submission
30. Wang P, Heber D, Henning SM. Quercetin increased the antiproliferative • Thorough peer review
activity of green tea polyphenol (-)-epigallocatechin gallate in prostate
• Inclusion in PubMed and all major indexing services
cancer cells. Nutr Cancer. 2012;64:580–7.
31. Datta SR, Ranger AM, Lin MZ, Sturgill JF, Ma YC, Cowan CW, Dikkes P, • Maximum visibility for your research
Korsmeyer SJ, Greenberg ME. Survival factor-mediated BAD phosphorylation
raises the mitochondrial threshold for apoptosis. Dev Cell. 2002;3:631–43. Submit your manuscript at
www.biomedcentral.com/submit