Heat shock protein 27 is a potential indicator for response to YangZheng XiaoJi and chemotherapy agents in cancer cells

Heat shock protein 27 is a potential indicator for response to YangZheng XiaoJi and chemotherapy agents in cancer cells

SIONED OWEN1,2* ,  HUISHAN ZHAO1-3* , ALWYN DART1,2 , YAmEI WANG1-3 ,     FIONA RUGE1,2 , YONG GAO4 ,  CONG WEI4,5 , YILING WU4-6  and WEN G . JIANG1,2

1Cardiff China medical Research Collaborative, Cardiff University School of medicine; 2Cardiff University-Capital medical

University Joint Centre for Biomedical Research, Cardiff University, Cardiff, UK; 3Cancer Institute and Key Laboratory

of Invasion and metastasis (Beijing), Capital medical University, Xitoutiao, Fengtai, Beijing; 4Yiling medical

Research Institute, Shijiazhuang; 5State Key Laboratory of Collateral Disease Research and Innovation medicine,

Shijiazhuang; 6Key Disciplines of State Administration of TCm for Collateral Disease, Shijiazhuang, Hebei, P.R . China

 

Received march 7, 2016;  Accepted April 22, 2016

DOI: 10.3892/ijo.2016.3685

 

 

Abstract. Heat shock protein 27 (HSP27) is a member of the heat shock protein family which has been linked to tumour progression and, most interestingly, to chemotherapy resis- tance in cancer patients . The present study examined the potential interplay between HSP27 and YangZheng XiaoJi, a traditional Chinese medicine used in cancer treatment. A range of cell lines from different tumour types including pancre- atic, lung, gastric, colorectal, breast, prostate and ovarian

cancer (both wild-type and resistant) were used . Levels and activation of HSP27 and its potential associated signalling pathways were evaluated by protein array and western blot- ting. Knockdown of HSP27 in cancer cells was achieved using siRNA. Localisation and co-localisation of HSP27 and other proteins were carried out by immunofluorescence. Cell growth and migration were evaluated in their response to a range of chemotherapeutic agents. The present study first identified, by way of protein array, that YangZheng XiaoJi was able to inhibit the phosphorylation of HSP27 protein in cancer cells . We further demonstrated that HSP27, which is co-localised with caspase-9, can be blocked from localising in focal adhesions and co-localising with caspase-9 by YangZheng XiaoJi. The study also demonstrated that YangZheng XiaoJi was able to sensitise cancer cells including those cells that were resistant to chemotherapy, to chemotherapeutic agents. Finally, knocking down HSP27 markedly reduced the migration of cancer cells

 

 

 

Correspondence to: Professor Wen G . Jiang, Cardiff China medical Research Collaborative, Cardiff University School of medicine, Henry Wellcome Building, Cardiff CF14 4XN, UK           E-mail: jiangw@cf.ac.uk                                                                      *Contributed equally

Key  words:  HSP27,  YangZheng  XiaoJi,  protein  kinase  array, caspases,  Focal  adhesion  kinase,  lung  cancer,  pancreatic  cancer, ovarian cancer, cellular migration

 

and increased the sensitivity of cancer cells to the inhibitory effect on cellular migration by YangZheng XiaoJi. YangZheng XiaoJi can act as an agent in first sensitising cancer cells to chemotherapy and secondly to overcome, to some degree, chemoresistance when used in an appropriate fashion in patients who have active HSP27.

Introduction

The heat shock proteins (HSPs) are ubiquitously expressed in almost all cells and across species. The protein family was so named owing to their initial discovery, namely, identified from cells under heat shock and is now known to be involved in a

number of other cellular stresses including heat, carcinogens, mechanical and chemical stress (1,2). These proteins serve as molecular chaperones and act as a mechanism to assist protein folding, repair damaged proteins, or assist in degrading the unwanted proteins after stress and injury, thus by doing so the proteins protect the injured cells from potentially lethal damage (3,4). The HSP proteins are thus important regula- tors in normal physiology, but are also involved in diseases including cardiovascular, wound healing and cancer.

Similarly discovered from cells under heat shock, HSP27 has been found to be widely involved in other types of cell stress, including oxidative response (5-8). The HSP27 is a protein of approximately 27 kDa in size and belongs to the HSP family in which sizes vary between 8-150 kDa . The main cellular function of HSP27 is protecting cells from becoming apoptotic, or anti-apoptotic. Although this role is critically important in homeostasis, it has serious implica- tions in clinical cancer. Firstly, HSP27 has been found to be overexpressed in a variety of human cancers and linked to a poor outcome for those cancer patients (9-13); secondly, high levels of HSP27 in cancer cells have been shown to promote cancer cell growth (14-16); and thirdly, HSP27 was found to be extremely elevated following chemotherapy, most probably as a protective mechanism to chemical stress (17-19). In the latter case, HSP27 and HSP70 were two of the gene products that were upregulated in a cisplatin resistant ovarian cancer cell line (19). Thus, this has caused an enormous challenge

 

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to cancer treatment and is frequently linked to chemotherapy

resistance in cancer.

The key mode of activation of HSP27 is by phosphoryla-

tion. Phosphorylation of HSP27 allows itself to form oligomers

which may facilitate the chaperone process under cellular

stress (20,21). Phospho-HSP27 interacts with other pro- apoptotic proteins such as DAXX and ASK1 and therefore by blocking pro-apoptotic protein interaction blocks the apoptotic pathway (22,23). Other known pathways of HSP27 include interaction with AKT1 and the oestrogen receptor. By inter- acting with these signalling proteins, phospho-HSP27 blocks some of their actions in cancer cells. The most commonly reported sites of phosphorylation with HSP27 are S15, S78,

S82 and S86. Consequently, phosphorylated HSP27 protein

levels have been found to be frequently raised in clinical

cancer tissues and chemoresistant cancer cells (24,25).

Together, it is suggested that HSP27 is a valid target for

cancer treatment. methods to either reduce the protein levels

of HSP27 or the phosphorylation of HSP27, would be desirable

in devising cancer treatment (26). A new anti-HSP27 agent,

known as OGX427 (Apatorsen) has entered into a late phase

clinical trial in patients with various malignant conditions

including pancreatic, lung, bladder or prostate cancer, alone

or in combination with other chemotherapies (27-30). Early

studies have shown a favourable outcome from the new treat-

ment.

There have been clinical trials which have shown that

YangZheng XiaoJi (YZXJ), a combination of traditional

Chinese medicinal herbs benefit cancer patients; however, how

this mechanism of action is achieved remains unknown (31-34).

The present  study reports  an  accidental  and  surprising

discovery that a traditional Chinese herbal medicine, known

as YangZheng XiaoJi (YZXJ) used in treating patients with

cancer, can suppress the phosphorylation of HSP27 and cell

functions related to this protein.

Materials and methods

 

Materials. Antibodies to human HSP27 (sc-13132), caspase-3

(sc-7148), caspase-8 (sc-70501), caspase-9 (sc-17784), phospho-

FAK (sc-81493), and GAPDH (sc-32233) were purchased from

Santa Cruz Biotechnologies Inc. (Santa Cruz, CA, USA).

Therapeutic agents including cisplatin, topotecan, pacilitaxol

and 5-FU were purchased from Sigma-Aldrich (Poole, Dorset, UK). Antibodies to FAK (ab131435) and phospho-HSP-27 (S86)

(ab17938) were purchased from Abcam (Cambridge, UK).

FITC/TRITC-conjugated Phalloidin and FITC- and TRITC

conjugated secondary antibodies were from Sigma-Aldrich.

Secondary antibodies (fluorescence- and HRT-conjugated)

were also from Sigma-Aldrich. Anti-HSP27 siRNA, control

siRNA and transfection reagents were also obtained from

Santa Cruz Biotechnologies Inc.

 

Cells. Human gastric cancer (AGS and HGC27), pancreatic

cancer (PANC1), ovarian cancer (SKOV3 and COV504), lung

cancer (A549 and SKmES1), breast cancer (mDA mB-231),

prostate cancer  (PC-3) cells were purchased from LGC Standard/ATCC (Southampton, UK). Ovarian cancer cells

A2780 and its cisplatin resistant strain A2780/CP70 were gifts

from Imperial College London (Dr Euan Stronach).

 

YangZheng XiaoJi extracts . An extract from YZXJ, named

DmE25 was prepared using a DmSO based method that has

been described in full (35,36). The herbal medicinal formula,

YangZheng XiaoJi was obtained from Yiling Pharmaceuticals

(Shijiazhuang,  HeBei,  China) .  The  formula  contained

the  following  16  ingredients:  Panax  ginseng  C .A .  Mey,

Astragalus memebranaceus  (Fisch .) Bge .var. mongholicus

(Bge .) Hsiao, Ligustrum  lucidum Ait,  Curcuma phaeocaulis

Val,  Ganodema  lucidum,  Gynostemma pentaphylla  (Thunb)

Mak,   Atractylodes   macrocephala   Koidz,   Scutellaria

barbata  D . Don,  Oldenlandia  diffusa  (willd .)  Roxb,  Poria

cocos,  Duchesnea  indica  Focke,  Solanum  lyratum  Thunb,

Artemisia scoparia (Bge.) Ki, Cynanchum paniculatum Kitag,

Eupolyphaga sinensis Walker, and Gallus domesticus Brisson.

For experimental use, the extract was diluted in the respective

cell culture media and the dilution range was between 1:100

to 1:2,000.

Immunofluorescent staining. Cells, plated with test agents in

8-well chamber slides, were first fixed using 4% formalin,

lightly permeabilised with 0.1% Triton X100 for 5 min and

blocked with a Tris buffer (25 mm, pH 8.4) that contained

7.5% pre-immuned goat serum for 1 h. Primary antibodies

(including anti-HSP27, anti-phospho-HSP27 (S86), anti-

caspases), diluted in the blocking buffer was added to the

respective slides which were kept in the dark at full humidity

on a slow moving platform for 1 h, and then washed thor-

oughly. FITC-tagged secondary antibodies were then added.

After 1 h, the slides were washed thoroughly and mounted

using FluorSave™ (Calbiochem, Nottingham, UK). TRITC-

conjugated phalloidin was diluted at final concentration of

10 µg/ml and added to the cells together with the secondary

antibodies. The slides were examined on an Olympus micro-

scope and photographed using a Hamamatsu digital camera.

The staining intensity was determined using ImageJ software.

Knockdown of HSP27. Cells at approximate 70% confluence

were rinsed with BSS buffer. Anti-HSP27 siRNA, diluted in

RNA free water was first mixed with a transfection reagent

and left at room temperature for 30 min before being added to

the cells. After 7 h medium was replaced and cells were left

to incubate for another 24 h. Expression of HSP27 was tested

using RT-PCR.

Cell growth assay. Cancer cells were first seeded in 96-well

tissue culture plates and allowed to adhere. The test materials

and their combinations were then added to the cells, which were

subsequently incubated for a period of 72 h. After removing the

culture media, cells were fixed with 4% formalin and stained

with 0.5% Crystal violet. After extensive washing, the staining

was extracted with 10% acetic acid and plates were read on

a multiple channel plate reader at 540 nm (Elx800; Bio-Tek, Swindon, UK). The test regimens included chemotherapeutic

agent alone, DmE25 alone, DmE25 and chemotherapeutic

agent combination and DmE25 pre-treatment followed by

chemotherapeutic agent.

Protein arraysfor detecting phosphorylation changes. Cancer

cells at 90% confluence in two T75 tissue culture flasks were

washed with BSS buffer and then placed into a fresh batch

 

Figure 1. (A) Detection of HSP27 and phospho-HSP27 in SKmES1 lung cancer cells after treatment with DmE-25 a YZXJ extract . (B) Changes in HSP27 and phospho-HSP27 in PANC-1 pancreatic cancer cells after treatment with DmE-25 a YZXJ extract.

 

 

 

of DMEM supplemented with 5% FCS. After 5 h, treatment

was added, again in 5% FCS. After a 5-h period, cells were

removed from the flasks with a cell scraper. The cells were

pelleted using a centrifuge at 2,500 rpm for 5 min. Lysis buffer was added to the cell pellets and placed on a spinning wheel for 1 h at 4˚C. The lysates were then spun at 12,000 x g for 10 min

at 4˚C . The supernatants were carefully collected and the

insolubles discarded. Based on absorbance the protein concen-

tration in the cell lysates were quantified using the scatter line

chart of microsoft Excel and then adjusted to 2 mg/ml. The

samples were stored at -20˚C until use. Antibody based protein

arrays, namely KAm850, which has 854 capture antibodies

spotted on to each array slides (Kinexus Bioinformatics

Ltd . , Vancouver, Canada) were used in the present study.

The following are the key parameters collected and used

for the data analysis: Globally Normalized Signal Intensity,

Background corrected intensity values are globally normal-

ized. The Globally Normalized Signal Intensity was calculated by summing the intensities of all the net signal median values

for a sample. Z Scores, Z score transformation corrects data

internally within a single sample. Z Score Difference, The

 

difference between the observed protein Z scores in samples

in comparison. Z Ratios, Divide the Z Score Differences by

the SD of all the differences for the comparison.

Electric  cell-substrate  impedance  sensing  (ECIS)  based

analyses on cell adhesion and cell migration . Briefly, 96-well

W96E1 microarrays were used on the ECIS Ztheta instrument

(Applied Biophysics Ltd., Troy, NJ, USA). Lung cancer cells

were added to the wells of the array, followed by immediate

tracking of cell adhesion over a range of frequencies (1,000

to 64,000 Hz) using automated modules. The adhesion was

analysed using the mathematical modelling methods as previ-

ously described. For cellular migration, confluent lung cancer

monolayers in the arrays were electrically wounded (2,000 mA

for 20 sec each), after which the migration of the cells was

immediately tracked, again over a range of frequencies. All the

experiments were conducted in triplicate.

Western blotting analysis  of HSP27 proteins . Proteins,

prepared in a similar fashion to that for protein analysis,

were separated on a SDS-PAGE genes, following transfer of

Figure 2. (A) Staining of phosphorylated HSP27 (S86) in SKmES1 human lung cancer cells (top panel) and pancreatic cancer PANC1 (bottom) after treat- ment with DmE-25. In the control, phospho-HSP27 was seen predominantly in the focal adhesion complex regions (white arrows). Treatment with YZXJ extract DmE25 resulted in almost complete loss of phospho-HSP27 from the focal adhesion complex (pink arrows). (B) Effects of DmE-25 on HSP27 and its activation in SKmES1 cells as shown by western blotting. (C) Effects of DmE-25 on HSP27 and its activation in PANC1 cells as shown by western blotting.

 

Table I . Effects of cisplatin on the growth of cancer cells in combination with DmE25 .

 

 

 

 

 

 

proteins to nitrocellulose membranes, the membranes were probed with the respective antibodies in a set protocol that including extensive washings in between the antibody probing and finally detecting the antibody signals using chemillumi- nescence reagents. The membranes were visualised using an imager (G-Box; Syngene, Cambridge, UK).

Results

DME25 has a marked effect on the phosphorylation of HSP27.

Using a protein array, we found that the YZXJ extract DmE25 resulted in a marked reduction of phosphorylation of HSP27,

 

particularly on Serine86 (S86) phosphorylation, in SKmES-1 lung cancer and PANC-1 pancreatic cancer cells (Fig. 1A and B, respectively).

Localisation of HSP27 and phospho-HSP27 (S86) in cancer

cells, assessed by  immunofluorescence .  HSP27  was  seen

broadly in the nucleus and in cytoplasmic region of lung cancer cells (Fig. 2A, top panel). It is very interesting to note that both total HSP27 and phospho-HSP27 was localised in focal adhesion and pseudopodia regions of the cells. Treatment of the lung cancer cells, SKmES1 with DmE25 resulted in loss of phospho-HSP27 from the focal adhesion and pseudopodia

Figure 3. (A) Co-staining phospho-HSP27 and caspase-9 in human lung cancer SKmES-1 cells. Control cells displayed high levels of staining of both phospho- HSP27 and caspase-9, both also showing a high degree of co-localisation (white arrows). However, the co-localisation was eliminated by DmE25 (bottom images). (B) Images under a higher power showing focal adhesion (FAC) and pseudopodia within the SKmES-1 cells.

Figure 4. (A) Co-staining phospho-HSP27 and phalloidin in human wild-type ovarian cancer A2780 (top panel) and A2780-CP70 (bottom panel) cells. (B) The co-localisation between phospho-HSP27 and caspase-9 was shown in the respective two cell lines A2780 (top panel) and A2780-CP70 (bottom panel). DmE25 is the extract from YZXJ.

regions of the cells, although the changes of total HSP27 did not appear to differ (Fig. 2). The same changes of phospho- HSP27 were seen in pancreatic cancer cells, PANC1 (Fig. 2A, bottom panel) as well as in ovarian cancer cell SKOV3 and gastric cancer AGS cells (data not shown). The inhibition on levels and activation of HSP27 was also demonstrated by western blotting analysis in both the lung and pancreatic cells (Fig. 2B and C, respectively).

Activated HSP27 is co -localised with caspase-9 in cancer

cells, which is prevented when cells are treated with DME25.

When co-stained for phospho-HSP27 (S86) and caspase-9, it was found that both molecules co-localised in regions of focal

 

adhesion (FAC) and pseudopodia (Fig. 3A and B). It was very interesting to note that when cells were treated with DmE25, this pattern of co-localisation appears to break (Fig. 3B).

Phospho-HSP27 and its co-localisation withfilamentous actin

and caspase-9 in cancer cells and in chemoresistant cancer cells. Using dual fluorescence staining, we have shown that phospho-HSP27 and F-actin did co-localise in FAC regions in both wild-type ovarian cancer A2780 (Fig. 4A, top panels) and in cisplatin resistant A2780/CP70 cells (Fig. 4 bottom panels). The same was seen with lung cancer cells (Fig. 5). In all the cells tested, treatment with DmE25 reduced the loca- tion of phospho-HSP27 to the FAC region. The co-localisation

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Table II . Effects of pacilitaxel on the growth of cancer cells in combination with DmE25 .

 

 

Figure 6. Wild-type A2780 and cisplatin resistance A2780/CP70 responded differently to cisplatin

of phospho-HSP27 and caspase-9 in wild-type A2780 and cisplatin resistant A2780/CP70 were also reduced in the pres- ence of DmE25 (Fig. 4B).

 

Effect of DME25 on the growth of ovarian cancer cells. A2780/

CP70 showed approximately 8 times less sensitivity to cisplatin compared with the wild-type parental cells (Fig. 6). Co-culture of both cisplatin and DmE25 resulted in the cells becoming more sensitive to cisplatin, as seen for lung, gastric, pancreatic and ovarian cancer cells (Tables I-V). However, knocking down HSP27 by siRNA did not significantly influence the sensitivity of the cells to the chemotherapeutic agents (data not shown).

HSP27  shows  marked  influence  on  cell  migration  and

acts synergistically with  YangZheng XiaoJi on cancer cell

migration . In light of the co-localisation of HSP27 and phal- loidin at the focal adhesion regions of cancer cells and our previous reports on the impact of DmE25 on the migration of various cells including cancer cells, we tested how HSP27 might affect cell migration. It was readily demonstrable that knocking down HSP27 resulted in a sustained reduction of cell migration (Fig. 7A and B). It was further confirmed that knocking down HSP27 increased sensitivity of cancer cells to low dosage chemotherapeutic agents and in particular when DmE25 was present (Fig. 7C and D).

Discussion

 

The current study stemmed from a surprising finding in our search for the potential pathways that are influenced by

INTERNATIONAL JOURNAL OF ONCOLOGY  49:  1839-1847,  2016                 

Figure 7. (A and B) Effects of knocking down HSP27 on the migration of SKmES1 cells. (C) The knockdown also increased the sensitivity of cancer cells to low concentration of drugs, in particular when combined with DmE25. (D) Effects of knocking down HSP27 on the migration of PANC-1 cells which is further decreased in the presence of DmE25.

 

Table III . Effects of 5-FU on the growth of cancer cells in combination with DmE25 .

 

 

 

 

 

DmE25, a traditional Chinese medicinal formula used in

cancer treatment.

HSP27, YangZheng XiaoJi and sensitivity to chemotherapeutic

agents. The role of HSP27 in the sensitivity to chemotherapy

agents is not without controversy. For example, it has been

reported recently that overexpression of HSP27 in pancreatic

cancer cells would increase the sensitivity of the cancer cells

to gemcitabine (37). Although reasons for this are not clear,

the type of drugs used in the study may be an explanation, as

these drugs are transported and act in cancer cells via different

mechanisms.

HSP27 acts as a cyto-protective molecule during androgen

independence in prostate cancer patients and in prostate

 

cancer cell models (38). Likewise, levels of HSP27 correlated

with resistance to irinotecan in colorectal cancer cells (39). HSP27, together with HSP70 is linked to the sensitivity of colorectal cancer to the naturally-occurring anticancer agent

curcumin (40).

Knocking down HSP27 sensitises glioblastoma multiforme

tumour cells to the HSP90 inhibitor,  17-N-allylamino-17-

demethoxygeldanamycin (17-AAG) and staurosporine by

changes in apoptosis (41). The same was seen in glioma cells,

in that silencing HSP27 resulted in an increase in apoptosis in

response to temozolomide and quercetin (42).

HSP27, YangZheng XiaoJi and cell migration . The finding that

HSP27 is co-localised with phalloidin to the focal adhesion

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Table IV. Effects of topotecan on the growth of cancer cells in combination with DmE25 .

 

Table V. Effects of cisplatin on the growth of wild-type and cisplatin-resistant ovarian cancer cells in combination with DmE25 .

 

 

 

regions of cancer cells is interesting. From the immunofluores- cence staining, HSP27 particularly the phosphorylated form of the protein, is frequently co-localised with Phallodin at the focal adhesion regions of cancer cells, where caspase-9 is also seen. It is of interest to note that inclusion of YZXJ extract DmE25 in the study markedly reduce the presence of phospho-HSP27 (S86) at the focal adhesions. Given the key role of focal adhesion in cell-matrix adhesion and cellular migration, it is plausible to suggest that activation of HSP27 (by way of phosphorylation) coordinates with other proteins in the focal adhesion area, such as integrins and the focal adhe- sion kinase (FAK) to regulate the adhesion and migration of cancer cells. We have recently reported that YZXJ was able to inhibit the phosphorylation of FAK in cancer cells and in endothelial cells (35,43).

Together with the finding that knocking down HSP27 resulted in an increase in cellular migration in cancer cells, it is suggested that HSP27, in particular when combined with YZXJ, is an important regulator of EmT reversal or mET. It has been reported recently that HSP27 is key to IL-6 dependent and IL-6 independent EmT in prostate cancer cells, by acting on IL-6 induced activation of STAT3/Twist and Snail (44,45). It is also involved in EGF induced EmT in prostate cancer cells by influencing the migration of these cells (46,47). Other than the reported effects on cancer cells, HSP27 has also been shown to be a mediator for angiogenesis (48), suggesting that HSP27 is involved in a wider range of processes in cancer development and progression.

Taken together, it is clear that HSP27 is a viable target in cancer treatment and that a combined approach, with either YZXJ or another method, would be a valid consideration when devising a therapy in either targeting HSP27 or in certain chemoresistance cases.

                                                                                                                                           SHOP NOW>>

Acknowledgements

The authors wish to thank Cancer Research Wales, National Research Network for Wales-Ser Cymru and the Albert Hung Foundation for supporting their work.

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