Caveolin-1 造成癌細胞多重抗藥性機制的探討

行政院國家科學委員會專題研究計畫 成果報告

Caveolin-1 造成癌細胞多重抗藥性機制的探討

中 華 民 國 94 年 10 月 30 日

Introduction:

Although chemotherapy has improved the prognosis of lung cancer patients, there

are still many patients who have initial resistance to chemotherapy or develop the

drug resistance after several courses of chemotherapy (1). Understanding and control

of drug resistance has become an important improvement in cancer therapy.

Caveolin-1, a 21- to 24-kD protein, is the principal component of caveolae, which

are special invaginated microdomains of the plasma membrane present in most

mammalian cells.(2) The role of caveolin-1 in cancer cells is quite diverse. Caveolin-1

functions as a tumor suppressor by contact inhibition of signaling molecules by its

scaffolding domain.(3) Up-regulation of caveolin-1 prevents anchorage-independent

growth (4) and sensitizes apoptotic machinery through phosphatidylinositol 3 kinase

(PI-3K) in different cells.(5) However, there is also substantial evidence that

caveolin-1 expression increases metastatic ability and increases the survival rate in

prostate cancer and other malignancies. (6, 7)

Recent studies have shown that acquisition of the MDR phenotype is associated

with the up-regulation of lipids including glucocylceramide (GlcCer) and cholesterol,

which constitute lipid rafts and caveolae. (8) Some multidrug resistant cancer cell

lines express very high caveolin-1 levels.(8-10) The up-regulation of caveolin-1 is

cancer cells.(9, 10) In addition, P-glycoprotein, a plasma membrane drug efflux

ATPase confering multidrug resistance, is enriched in caveolae within some MDR

cancer cells. However, caveolin-1 expression is not necessarily correlated with

P-glycoproein, because caveolin-1 is up-regulated in MDR cells with little

P-glycoprotein expression.

Cisplatin based combination chemotherapy displays significant anti-tumor activity

against lung cancer and remains one of the regimens in lung cancer patients. Although

caveolin-1 is over-expressed in cisplatin treated cancer cells compared with parent

cells, there is no direct correlation between caveolin-1 expression and cisplatin

resistance. In this study, we observe the cavoelin-1 and P-glycoprotein expression in

the paraffin-embedded specimens from pathologically proven lung cancer patients

who received cisplatin plus gencitabine after biopsy. Correlation between caveolin-1

expression and the chemotherapy resistance is established.

Materials and Methods:

Cell Culture

Cells were grown in Dulbecco’s modified Eagle’s medium supplemented with

10% heat-inactivated fetal bovine serum and 2 mM L-glutamine at 37oC, 5% CO2 in a

humidified incubator. Different concentrations of cytotoxic drugs such as cisplatin

added in the culture medium.

An SV-40-transformed, nontumorigenic bronchial epithelial cell line, BEAS-2B

(CRL9609; American Type Culture Collection, Rockville, MD), was grown in

modified F12 medium admixed with Hepes stock solution (1.5 mol/L, pH 7.2 to 7.4)

and supplemented with growth factors as previously described.

SDS-PAGE and Western Blotting

The cells (1x106) were harvested and prepared by application of 500 µl of boiling

2X concentrated electrophoresis sample buffer (125 mM Tris-HCl, pH 6.8, 2% SDS,

5% glycerol, 0.003% bromophenol blue, and 1% β-mercaptoethanol) to each 10-cm

diameter dish. Protein samples were separated by SDS-PAGE and transferred to a

PVDF membrane, which was subjected to immunoblotting by anti-human caveolin-1

antibody (1:1000 dilution, BD Transduction Laboratories, Lexington, KY), followed

by blotting with HRP-conjugated rabbit anti-human antibody (1: 3500 dilution,

Amersham, Buckinghamshire, United Kingdom). An enhanced chemiluminescence

(ECL) reaction (Amersham) was applied for signal detection. Immunoblotting with

anti-human α-tubulin antibody (1:1000 dilution, Oncogene, Darmstadt, Germany)

was used as an internal marker both for quantity and quality control.

Immunohistochemistry

diagnosed as non-small cell lung cancer were obtained from the Department of

Pathology, National Taiwan University Hospital, during the period of 1998 to 2000.

Tumor staging was performed using the TNM classification system of international

union against cancer. The follow-up data was obtained from medical records. All

patients had advanced diseases, at least stage IIIb, in the initial presentation and did

not receive total excision for the tumor. They all had good performance and received

chemotherapy with the regimen of gencitabine plus cisplatin after diagnosis.

Immunohistochemistry

Paraffin-embedded 5 µm-thick sections were deparaffinized, heated in citrate

buffer (0.01M), treated with 0.3% H2O2, and re-hydrated. After blocking, serial

sections from the same patients were incubated respectively with anti-caveolin-1 IgG

(clone 2297, 1:500 dilution, BD Transduction Laboratories, Lexington, KY) and

anti-P-glycoprotein IgG (clone C494, 1:250 dilution, Signet Laboratories, Dedham,

MA) for 1 hour at room temperature, washed, and then blotted with biotin-labeled

bridge antiserum (1:200, Vector Laboratories, Burlingame, CA). After several washes

with PBS, sections were incubated with a solution of avidin and biotin-conjugated

peroxidase complex (Vector Laboratories) for 30 minutes at room temperature. DAB

colorization was applied, and slides were further counter-stained with hematoxylin.

For each specimen, the entire population of cancer cells in the lung tissue was

scanned. All specimens were evaluated without knowledge of the patients’ clinical

data. Caveolin-1 immunoreactivity was normally localized to fibroblasts, type I

pneumocytes, and endothelial cells of blood vessels in all tissue specimens examined,

which served as an internal quality control in immunohistochemistry. We artificially

chose 30% caveolin-1 immunoreactivity as a cutoff value for the assignment of

positivity (or negativity) of caveolin-1 staining. Although antibody against

P-glycoprotein had shown to cross-react with Pyruvate Carboxylase, unequivocal plasma

membrane patterns of immunostaining represent true P-glycoprotein expression. Positive staining of P-glycoprotein was defined if 30% or more of the neoplastic cells showed membrane staining.

Statistical Analysis

The correlation of caveolin-1 immunoreactivity with patients’ clinical variables was

analyzed by χ2-test or Fisher’s exact test (2-sided). Survival curve was plotted by

the Kaplan-Meier product-limit method and the statistical significance was assessed

by the log-rank test. Univariate and multivariate analyses were performed using the

Cox proportional-hazards regression model. P < 0.05 was considered to indicate

statistical significance in all of the analyses. All analyses were done with statistical

Result:

Up-regulation of caveolin-1 expression by cytotoxic drugs in drug-sensitive

cancer cells

We treated the lung cancer cell lines A549 with cisplatin and gemcitabine cytotoxic

drugs. As shown in Figure 1, drug-sensitive A549 cells had less or absent caveolin-1

expression. After exposure of the cancer cells to these cytotoxic drugs, up-regulation

of caveolin-1 expression was found. Higher concentration of cytotoxic drug and

longer exposure time also increased the expression of caveolin1.

Clinical Finding

We examined the status of caveolin-1 expression in specimens obtained from

patients who received chemotherapy. In this retrospective study, 73 specimens

diagnosed as lung cancer were obtained from the Department of Pathology, National

Taiwan University Hospital, during the period of 1998 to 2000. All patients had

advanced diseases, good performance and received chemotherapy with the regimen of

gencitabine plus cisplatin. The patients consisted of 41 man and 32 women. The mean

age was 57 years old (range 25-83). Forty-four patients had an age lower than 65

years old with 29 patients older than 65 years old. The pathological findings disclosed

adenocarcinoma in 49 patients, squamous cell carcinoma in 18 patients, and large cell

classification of the International Union against cancer. Eleven patients were in stage

IIIb, and 59 patients were in stage IV. Twenty-three patients had response to

chemotherapy, 32 patients remained stable during chemotherapy and 19 patients had

disease progression during chemotherapy.

Expression of cavoelin-1 in advanced lung cancer

Caveolin-1 immunoreactivity was normally localized to fibroblasts, type I

pneumocytes, and endothelial cells of blood vessels in all tissue specimens examined,

which could serve as an internal quality control in immunohistochemistry. We

artificially chose 30% caveolin-1 immunoreactivity as a cutoff value for the

assignment of positivity (or negativity) of caveolin-1 staining. Twelve of 73 patients

had caveolin-1 expression (Fig. 2). To determine the significance of caveolin-1

expression, all patients were divided into two groups according their cavoelin-1

expression: caveolin-1 negative group (n=61 patients) and cavoelin-1 positive group

(n=12 patients). The clinicopathologic variables of the caveolin-1 negative and

caveolin-1 positive group are listed in table 1.

Correlation of caveolin-1 expression and poor response to chemotherapy

Among the 12 patients with caveolin-1 expression, only four patients remained

stable disease during chemotherapy. The other eight patients had progressive disease

caveolin-1 expression found in their specimens. Correlation between caveolin-1

expression and progress disease during chemotherapy was established (P=0.01, table

1).

We also examined the status of P-glycoprotein expression in these specimens and

4 patients had unequivocal plasma membrane patterns of immunostaining represent true

P-glycoprotein expression. In these 4 patients, one patient had progress disease during chemotherapy, two patients had partial response during chemotherapy and one patient remained stable during chemotherapy. Only one patient had caveolin-1 and P-glycoprotein co-expression and this patient remained stable during chemotherapy.

Prognostic significance of caveolin-1 expression

Analysis of the association of caveolin-1 expression with patients’ survival rate

was performed on the 73 patients of non-small cell lung cancer. The survival time

after chemotherapy for these 12 patients with caveolin-1 expression and 61 patients

without caveolin-1 expression was calculated by the Kaplan-Meier method (Fig. 3).

Multivariate analysis was also performed for caveolin-1 immunoreactivity and other

factors as stage, age, sex, and chemotherapy response. Performance status was

excluded for analysis because all of the patients were good enough to received

chemotherapy at the beginning. The univariate log-rank test for each variable was

multiple cog regression model suggested that the expression of caveolin-1 was an

independent factor for prediction of poor survival in patients with non-small cell lung

cancer (hazard ratio 3.516; P=0.0008) (Table 2)

Discussion:

In this study, immunohistochemical staining was performed in 73 specimens

from lung cancer patients with advanced disease receiving chemotherapy. Only 12

patients had caveolin-1 immunostaining. Among them, four patients had stable

disease during chemotherapy and eight had disease progression during chemotherapy.

Caveolin-1 expression is correlated with a poor response to chemotherapy. Although

up-regulation of caveolin-1 was found in many MDR cancer cell lines (8-11), this is

the first study with clinical data to demonstrate the relationship between caveolin-1

expression and possible drug resistance.

Caveolin-1 is thought to be a tumor suppressor gene, and its expression is

down-regulated in oncogenically transformed fibroblast, as well as breast and lung

cancer (12-14). However, after cytotoxic drug stress, up-regulation of caveolin-1

could be seen both in cancer lines and in vivo (15, 16). It has been reported that little

or no caveolin-1 immunostaining is in non-small cell lung cancer patients before

chemotherapy. But re-expression of caveolin-1 has been observed in half of the

Up-regulation of caveolin-1 after cytotoxic drugs is thought to be an early cellular

response before drug resistance is manifested. However, in this study, 12 of 73

patients who had advanced non-small cell lung cancer (at least stage IIIb) without

chemotherapy before biopsy showed positive caveolin-1 immunostaining. Many of

them had disease progression during chemotherapy. Expression of caveolin-1 before

administration of cytotoxic drugs is a good predictor for drug resistance.

Several mechanisms have been proposed for caveolin-1 up-regulation in MDR

cells. P-glycoprotein (P-gp), an ATPase that pumps out drugs used in chemotherapy,

has been found to be enriched in caveolin-1 rich membrane domain in some MDR

cells (9). But up-regulation of caveolin-1 is correlated with little P-gp expression in

other MDR cells and over-expression of P-gp is not localized in caveolae (17).

Reintroduction of caveolin-1 expression into drug sensitive cancer cells does not

increase the expression of P-gp (9, 18). In the specimens of ovarian cancer patients,

caveolin-1 expression is not correlated with P-gp expression (19). In our study,

P-glycoprotein was present in 4 patients without correlation of caveolin-1 expression.

It is interesting that caveolin-1 and cholesterol could regulate each other

reciprocally (20-23). Induction of caveolin-1 expression enhances the cholesterol

efflux and reduction of their expression with caveolin-1 antisense DNA, oxysterols or

Caveolin-1 is also transcriptionally regulated by

sterol-regulatory-element-binding-protein (SREBP) (20). A high cholesterol efflux

pathway may serve as a platform for lipophilic drugs transport from intracellular

compartment to plasma membrane against a steep concentration gradient, although

there is still no direct evidence to elucidate this. However, some cytotoxic drugs used

to establish MDR cancer cell lines with caveolin-1 up-regulation have low

lipophilicity including doxorubicin and cisplatin.

Another possibility is that caveolin-1 expression exerts negative regulation in

apoptotic machanisms, although such results are in recent conflict. Down-regulation

of caveolin-1 by anti-sense vector prevents staurosporine-induced apoptosis in

NIH-3T3 and T24 cells (25). Caveolin-1 sensitizes Rat-1 fibroblast to

ceramide-induced apoptosis and also increases 293 cells and HeLa cells to arsenite

cytotoxicity through PI-3K pathway (5, 26). However, Caveolin-1 could inhibit

anoikis in MCF-7 breast cancer cells (27), and suppresses c-myc-induced apoptosis

(28). In acid sphingomyelinase-deficient (asmase-/-_{) mice, loss of caveolae and}

glycolipid-enriched membrane microdomains (GEM) increases susceptibility to

apoptosis after T cell receptor stimulation probably through decrease of MAPK

activation (29). A recent paper reported caveolin-1 has anti-apoptotic effect in prostate

and MDM2 (30). These conflicting reports may indicate caveolin-1 effect are cell

type- and expression level-dependent (5, 28, 31).

In summary, our study reveals that up-regulation of caveolin-1 in cancer cells has

more cytotoxic drug resistance no matter hydrophilic or lipophilic drugs. Caveolin-1

is down-regulated in newly-transformed cells and almost absent in the early stages of

lung cancer (14). However, its re-expression in the late stage disease before

chemotherapy is correlated with the drug resistance. Caveolin-1 may be one

component of mechanisms in multidrug resistance and may serve as a therapeutic

target for preventing multidrug resistance.

Reference:

1. Nishio, K., Nakamura, T., Koh, Y., Suzuki, T., Fukumoto, H., and Saijo, N.

Drug resistance in lung cancer. Curr Opin Oncol, 11: 109-115, 1999.

2. Anderson, R. G. W. and Jacobson, K. A Role for Lipid Shells in Targeting

Proteins to Caveolae, Rafts, and Other Lipid Domains. Science, 296:

1821-1825, 2002.

3. Engelman, J. A., Chu, C., Lin, A., Jo, H., Ikezu, T., Okamoto, T., Kohtz, D. S.,

and Lisanti, M. P. Caveolin-mediated regulation of signaling along the p42/44

MAP kinase cascade in vivo. A role for the caveolin-scaffolding domain.

4. Engelman, J. A., Wykoff, C. C., Yasuhara, S., Song, K. S., Okamoto, T., and

Lisanti, M. P. Recombinant Expression of Caveolin-1 in Oncogenically

Transformed Cells Abrogates Anchorage-independent Growth. J. Biol. Chem.,

272: 16374-16381, 1997.

5. Zundel, W., Swiersz, L. M., and Giaccia, A. Caveolin 1-mediated regulation of

receptor tyrosine kinase-associated phosphatidylinositol 3-kinase activity by

ceramide. Mol Cell Biol, 20: 1507-1514, 2000.

6. Podar, K., Tai, Y. T., Cole, C. E., Hideshima, T., Sattler, M., Hamblin, A.,

Mitsiades, N., Schlossman, R. L., Davies, F. E., Morgan, G. J., Munshi, N. C.,

Chauhan, D., and Anderson, K. C. Essential role of caveolae in interleukin-6-

and insulin-like growth factor I-triggered Akt-1-mediated survival of multiple

myeloma cells. J Biol Chem, 278: 5794-5801, 2003.

7. Wu, D., Foreman, T. L., Gregory, C. W., McJilton, M. A., Wescott, G. G., Ford,

O. H., Alvey, R. F., Mohler, J. L., and Terrian, D. M. Protein kinase cepsilon

has the potential to advance the recurrence of human prostate cancer. Cancer

Res, 62: 2423-2429, 2002.

8. Lavie, Y., Fiucci, G., and Liscovitch, M. Upregulation of caveolin in multidrug

resistant cancer cells: functional implications. Adv Drug Deliv Rev, 49:

9. Lavie, Y., Fiucci, G., and Liscovitch, M. Up-regulation of caveolae and

caveolar constituents in multidrug-resistant cancer cells. J Biol Chem, 273:

32380-32383, 1998.

10. Yang, C. P., Galbiati, F., Volonte, D., Horwitz, S. B., and Lisanti, M. P.

Upregulation of caveolin-1 and caveolae organelles in Taxol-resistant A549

cells. FEBS Lett, 439: 368-372, 1998.

11. Lavie, Y. and Liscovitch, M. Changes in lipid and protein constituents of rafts

and caveolae in multidrug resistant cancer cells and their functional

consequences. Glycoconj J, 17: 253-259, 2000.

12. Galbiati, F., Volonte, D., Engelman, J. A., Watanabe, G., Burk, R., Pestell, R.

G., and Lisanti, M. P. Targeted downregulation of caveolin-1 is sufficient to

drive cell transformation and hyperactivate the p42/44 MAP kinase cascade.

Embo J, 17: 6633-6648, 1998.

13. Lee, S. W., Reimer, C. L., Oh, P., Campbell, D. B., and Schnitzer, J. E. Tumor

cell growth inhibition by caveolin re-expression in human breast cancer cells.

Oncogene, 16: 1391-1397, 1998.

14. Ho, C. C., Huang, P. H., Huang, H. Y., Chen, Y. H., Yang, P. C., and Hsu, S. M.

Up-regulated caveolin-1 accentuates the metastasis capability of lung

1647-1656, 2002.

15. Belanger, M. M., Roussel, E., and Couet, J. Up-regulation of caveolin

expression by cytotoxic agents in drug-sensitive cancer cells. Anticancer

Drugs, 14: 281-287, 2003.

16. Belanger, M. M., Roussel, E., and Couet, J. Caveolin-1 Is Down-Regulated in

Human Lung Carcinoma and Acts as a Candidate Tumor Suppressor Gene.

Chest, 125: 106S-, 2004.

17. Hinrichs, J. W., Klappe, K., Hummel, I., and Kok, J. W. ATP-binding cassette

transporters are enriched in non-caveolar detergent-insoluble

glycosphingolipid-enriched membrane domains (DIGs) in human

multidrug-resistant cancer cells. J Biol Chem, 279: 5734-5738, 2004.

18. Liscovitch, M. and Lavie, Y. Multidrug resistance: a role for cholesterol efflux

pathways? Trends in Biochemical Sciences, 25: 530-534, 2000.

19. Davidson, B., Goldberg, I., Givant-Horwitz, V., Nesland, J. M., Berner, A.,

Bryne, M., Risberg, B., Kopolovic, J., Kristensen, G. B., Trope, C. G., van de

Putte, G., and Reich, R. Caveolin-1 expression in ovarian carcinoma is MDR1

independent. Am J Clin Pathol, 117: 225-234, 2002.

20. Bist, A., Fielding, P. E., and Fielding, C. J. Two sterol regulatory element-like

low density lipoprotein free cholesterol. Proc Natl Acad Sci U S A, 94:

10693-10698, 1997.

21. Fielding, P. E. and Fielding, C. J. Plasma membrane caveolae mediate the

efflux of cellular free cholesterol. Biochemistry, 34: 14288-14292, 1995.

22. Fielding, C. J., Bist, A., and Fielding, P. E. Caveolin mRNA levels are

up-regulated by free cholesterol and down-regulated by oxysterols in

fibroblast monolayers. Proc Natl Acad Sci U S A, 94: 3753-3758, 1997.

23. Fielding, C. J., Bist, A., and Fielding, P. E. Intracellular cholesterol transport in

synchronized human skin fibroblasts. Biochemistry, 38: 2506-2513, 1999.

24. Fielding, C. J. and Fielding, P. E. Intracellular cholesterol transport. J Lipid

Res, 38: 1503-1521, 1997.

25. Liu, J., Lee, P., Galbiati, F., Kitsis, R. N., and Lisanti, M. P. Caveolin-1

expression sensitizes fibroblastic and epithelial cells to apoptotic stimulation.

Am J Physiol Cell Physiol, 280: C823-835, 2001.

26. Shack, S., Wang, X. T., Kokkonen, G. C., Gorospe, M., Longo, D. L., and

Holbrook, N. J. Caveolin-induced activation of the phosphatidylinositol

3-kinase/Akt pathway increases arsenite cytotoxicity. Mol Cell Biol, 23:

2407-2414, 2003.

anchorage-independent growth, anoikis and invasiveness in MCF-7 human

breast cancer cells. Oncogene, 21: 2365-2375, 2002.

28. Timme, T. L., Goltsov, A., Tahir, S., Li, L., Wang, J., Ren, C., Johnston, R. N.,

and Thompson, T. C. Caveolin-1 is regulated by c-myc and suppresses

c-myc-induced apoptosis. Oncogene, 19: 3256-3265, 2000.

29. Nix, M. and Stoffel, W. Perturbation of membrane microdomains reduces

mitogenic signaling and increases susceptibility to apoptosis after T cell

receptor stimulation. Cell Death Differ, 7: 413-424, 2000.

30. Li, L., Ren, C. H., Tahir, S. A., Ren, C., and Thompson, T. C. Caveolin-1

maintains activated Akt in prostate cancer cells through scaffolding domain

binding site interactions with and inhibition of serine/threonine protein

phosphatases PP1 and PP2A. Mol Cell Biol, 23: 9389-9404, 2003.

31. Li, L., Yang, G., Ebara, S., Satoh, T., Nasu, Y., Timme, T. L., Ren, C., Wang, J.,

Tahir, S. A., and Thompson, T. C. Caveolin-1 mediates testosterone-stimulated

survival/clonal growth and promotes metastatic activities in prostate cancer

cells. Cancer Res, 61: 4386-4392, 2001.

Western blotting revealed caveolin-1 expression in BEAS-2B cells, but little

expression in A549. After exposure to different cytotoxic drugs including cisplatin

and gemcitabine, up-expression of caveolin-1 in A549 was found. Expression of

caveolin-1 was higher in high dose cytotoxic drug and longer exposure time. Staining

of α-tubulin was used as control.

Figure 2.

Immunohistochemical staining for caveolin-1. A. Negative or faint staining of

caveolin-1 in tumor cells from the patient who was response to chemotherapy.

Positive staining of endothelial cells as internal control (arrow). B. Cancer cell

chemotherapy.

Figure 3.

Comparison of overall survival curves for patients with caveolin-1-positive and

Table 1. Relationship between Clinicopathologic Features and Caveolin-1 Expression in Biopsy Specimens of Non-small Cell Lung Cancer

Characteristic Caveolin-1 (-) No. of Patients (%) Cavelin-1 (+) No. of Patients (%) P value Age 57.9± 13 62.7 ± 14 0.14 Gender Male 34 (82.9) 7 (13.1) 1 Female 27 (84.4) 5(15.6) Stage Ⅲb 11 (91.7) 1 (8.3) 0.68 Ⅳ 50 (82) 11 (18) Cell Type Adenocarcinoma 39 (84.8) 7 (15.2) 0.75 Others 22 (81.5) 5(18.5) Chemotherapy response Progress disease 12 (63.2) 7(36.8) 0.01§ Others 49 (90.7) 5(9.3) § _Significance

Table 2. Analysis of parameters including caveolin-1 immunoreactivity and other clinical prognostic factors in 73 patients with advance lung cancer

Cog Regression

Factors

Hazard Ratio(95%CIa₎

P

a _{CI, confidence interval} b _{Cut-off value: 30%}

c _{Adenocarcinoma vs. non adenocarcinoma}