Abstract
OBJECTIVE To investigate the relationship between the level of caspase-3 and 8 expression and intracellular Ca2+ levels in BCML-TA299 breast cancer cells in the process of apoptosis.
METHODS Mice were divided into three IFNa-treated groups and one control group as follows: an intratumoral injection, subcutaneous injection, preventive injection, and a control without injection. The cellular DNA content, changes in the cell cycle and the relationship between the cell Ca2+ concentrations and the expression of caspase-3 and 8 were examined.
RESULTS After injection of IFN-α-2b by different routes, the morphologic transformation of the breast cancer cells in each group was observed. There was a typical apoptotic response in the intratumoral-injection group. The expression of caspase-3 and 8 was diverse among the experimental groups, and correlated with the cellular Ca2+ concentration. Caspase-3 and 8 expression and the cellular Ca2+ level were higher following intratumoral injection compared to the other treatments (P<0.01). Among the experimental groups, the cell cycle displayed definitive changes.
CONCLUSION a) Both caspase-3 and 8 and the intracellular Ca2+ are elevated in the process of cell apoptosis in BCML-TA299 breast cancer tissues. These changes may play important roles in the occurrence and development of breast cancer; b) Variation in the route of IFN-α-2b administration can produce different responses in the expression of caspase-3 and 8 and the concentration of Ca2+ in apoptotic tumor cells.
keywords
At present, investigations of signaling transduction in apoptotic cells involve many aspects, such as Ca2+ concentration, intracellular cAMP, oxidative stress, protein kinases, protein phosphorylation and dephosphorylation etc. Most results have shown that Ca2+, as an important signaling transduction factor, can activate the caspase cascade, and thus take part in abnormal apoptosis in tumors[1,2]. We induced apoptosis in breast cancer (BCML-TA299) cells by IFN-α-2b, set up an apoptotic model and investigated the correlation between changes of Ca2+ concentration and the expression of caspase-3 and 8. By this study we may find new clues or therapeutic targets against breast cancer.
MATERIALS AND METHODS
Materials
Main reagents and instruments: IFN-α-2b was purchased from the Tianjin HuaLi Da Biotechnology Co, Ltd. A SABC immunohistochemistry kit for caspases-3 and 8, PBS buffer and DBA color reagent were purchased from the Tianjin HaoYang Biotechnology Co, Ltd. A Coulter EPICS-XL EXPOII flow cytometer was purchased from the USA.
Tumor strains: BCML-TA299 breast cancer cells were obtained from the experimental tumor laboratory in the Tianjin Cancer Institute affiliated with Tianjin Medical University.
Experimental animals: Healthy, 8-week-old TA2 female mice weighing 18-20 g were purchased from the animal laboratory of the Tianjin Cancer Institute.
Methods
Tumor transplants
Tumor-bearing mice were killed by cervical dislocation and the isolated tumor tissue was cut into 2 mm3 pieces. A single tissue piece was then transplanted into the left mammary region of each experimental mouse.
IFN-α-2b administration
Dosages of IFN-α-2b for mice were based on the clinical therapeutic dosages for patients[3] and weight of the mice: 5 U/0.1ml injected once every two days for a total of 20 days. Flou-3 Am ester and F-127 were prepared following directions in the kit. Flou-3 Am ester(0.1mg) was dissolved in 89 ul DMSO.
Experimental groups
Sixty mice were randomly divided into 4 groups with 15 mice/group: 1)a intratumoral-injection group received IFN-α-2b in the carcinoma tissue when the tumor diameter was over 0.6-0.8 cm; 2)a subcutaneous-injection group was treated with IFN-α-2b at a site distant from the transplant on the second day after transplantation; 3)a preventive-injection group received subcutaneous IFN-α-2b twice before transplantation; 4) a control group received no interventions.
Analysis of Ca2+ concentration using FCM
The carcinoma tissue was isolated from the mice 24 h after the last IFN-α-2b administration. Then a monocellular suspension was made and adjusted to 1×106cells/L, after which 1 μl F-127 and Flou 3/Am was added to produce a final concentration of 20 μmol/L. The cells were incubated for 30 min at 37°C in a water bath, centrifuged, washed three times with serumfree-culture solutions and the cellular fluorescence measured by FCM.
Analysis of cellular DNA content and apoptosis percentage using FCM
The collected cells were fixed with 75% cold alcohol at 4°C overnight, washed twice with PBS buffer, adjusted to 1×106cells/L, stained with PI and analyzed by FCM.
Immunohistochemical (SABC) detection of caspase-3 and 8
The breast cancer tissue from different groups was isolated and fixed with 10% formalin, embedded with paraffin and cut into consecutive sections. After routine deparaffination and hydration, endogenous perxisome activity was blocked and the cells were washed in PBS twice for 5 min each time. The antigens were unmasked by microwave treatment and the slides incubated with primary antibodies at 4°C overnight. Other steps were conducted according to the instructions of the SABC kit. Positive control sections were supplied by the Tianjin HaoYang Biotechnology Co. Ltd, and PBS was used in place of antibodies as negative control. DAB coloration was developed and the cells restained with H&E.
Criteria for evaluation of the results
Cells with brown granules or diffuse lamella more than 10% in the cytoplasm were positive for caspase-3 or 8. A region with clearly labeled and uniform distribution was selected and the positive cells in a 400×field counted. Ten fields for each sample were counted and the mean value used as the cell apoptotic index.
Statistics
SPSS10.0 statistical software package was used to analyze the data and oneway ANOVA was used to compare the variance among the four groups. The Q-test was employed to determine the difference between any of the two groups.
RESULTS
Cell morphologic transformation after administration of IFN-α-2b by different routes
Compared with the control group, mice bearing breast cancer cells treated with IFN-α-2b showed focal necrosis at different levels, intact membranes, protuberant nuclei, condensed chromatin and apoptotic bodies. Some regions manifested cellular edema, pyknosis, karyorrhexis and karyolysis. These presentations were especially obvious in the intratumoralinjection group(Fig.1A), while cancer cells of the control group grew actively and showed caryocinesia in some regions(Fig.1B).
H&E. staining results of the BCML-TA299 breast tumor tissues. A: control group; cancer cells grew actively, arrayed like a glandular tube. There were abundant small blood vessel among the cancer cells, and evidence of cellular proliferation. B: intratumoral-injection group; cancer cells became degenerative and necrotic, showing karyopyknosis. C: subcutaneous-injection group; cancer cells became degenerative and necrotic, there were vacuoles in the cell nuclei, and an indication of karyopyknosis and karyorrhexis. D: preventive-injection group; cancer cells became degenerative and necrotic, and showed karyopyknosis (Magnification: ×400).
Analysis of Ca2+ levels with FCM after different routes of IFN-α-2b treatment
The level of Ca2+ concentrations in the control group, intratumoral-injection group, subcutaneous-injection group and preventive-injection group were 61.7 nmol/L, 1,413.4 nmol/L, 20.5 nmol/L and 2.0 nmol/L respectively. Compared with the control group, experimental groups displayed a significant difference (P<0.01, Table 1).
Intracellular Ca2+ levels of the experimental groups.
The expression of caspase-3 and 8 in different experimental groups
Compared with the control group(Fig. 2B;Fig. 3B), the expression of caspase-3(Fig.2) and 8(Fig.3) was significantly activated in the intratumoral-injection group but not in the subcutaneous-injection group or preventive-injection group. There was a significant difference between each experimental group compared to the control group (Table 2).
The expression of caspase 3 in breast cancer tissue after different routes of IFN-α-2b administration. A: control group; B: intratumoral-injection group; C: subcutaneous-injection group; D: preventive-injection group (SABC, ×400).
The expression of caspase 8 in breast cancer tissue after different routes of IFN-α-2b administration. A: control group; B: intratumoral-injection group; C: subcutaneous-injection group; D: preventive-injection group (SABC, ×400).
The expression of caspase-3 and 8 in different groups.
Analysis of DNA content and apoptotic rate with FCM
In the G0~G1 transition, there were hypodiploid cells (apoptotic peak). After administration by different routes, the apoptotic index obviously increased in the experimental groups, especially in the intratumoralinjection group (Table 3).
Cell cycle of the experimental groups.
DISCUSSION
Apoptosis is considered to be a physical cell-death process, strictly regulated by polygenes, which play important roles in the biological development and maintenance of cell self-homeostasis. At present, some studies have demonstrated that extracellular sig nals and intracellular second messengers can induce cell apoptosis[4~6].
Apoptosis can be divided into 3 stages: signal transduction, central regulation and structural transforma tion. Transduced exogenesis and endogenesis death signals result in activation of caspase protein kinase leading to protein substrate cleavage and disorganization of the cells[7~9]. In addition, some research has demonstrated that free cytoplasmic Ca2+ concentration increases instantly, and is maintained at the initial stage of apoptosis followed by activation of endonuclease, DNA degradation and cell death. Therefore there is reason to believe that Ca2+ influx and a fall in the mitochondria membrane potential are at the upper stream of the caspase cascade reaction[10].
Because IFN-α-2b is a commonly used therapeutic agent in clinics, we chose IFN-α-2b to induce apotosis resulting in a typical apoptotic response: 1) Cell morphologic transformation showed that the cells became round, membranes became protuberant, nuclei condensed and apoptotic bodies formed. 2)On the left of the G1 phase of Fig.4, hypodiploid cells revealed an apoptotic peak. We examined Ca2+, caspase-3 and 8 as part of the apoptotic signaling transduction pathway in BCML-TA299 breast cancer cells. Results revealed that there were different changes in Ca2+ concentration with different routes of IFN-α-2b administrations. For example the cellular Ca2+ concentration in the intratumoral-injection group was 22 times higher than the control group. In addition, growth of the cancer was significantly blocked at the G0~G1 transition, suggesting that Ca2+ was of importance to trigger and transducte apoptotic signals. Under normal physiological conditions, intracellular and extracellular Ca2+ levels can maintain a steady dynamic balance, while our research showed that the Ca2+ concentration in the control and preventive groups was obviously lower than the intratumoral injection group. With regard to this phenomenon some researchers[11] think the decrease of Ca2+ concentration in the cells can also cause cell apoptosis, which may be related to the decrease in ATP concentration due to its use in pumping Ca2+ from the endoplasmic reticulum Ca2+ pool. Because carcinoma cells show abnormal proliferation with little apoptosis, the means to accelerate cell apoptosis by activating the caspase protein kinase system is an important goal. Some reports have indicated that caspase 8 acts as a trigger in a cascade reaction. When activated, caspase 8 may induce death-signaling complex formation and further activate caspase 3, which is the last step, leading to activation of an endonuclease, DNA degradation and apoptosis[12~14].
FCM analysis of the cell cycle of breast cancer cells after different routes of IFN-α-2b administration. A: control group; B:intratumoral-injection group; C: subcutaneous-injection group; D: preventive-injection group.
We investigated apoptosis in BCML-TA299 breast cancer cells after injecting IFN-α-2b by different routes, and detected the expression of caspase 3 and 8 by SABC. The results showed that intratumoral injections resulted in increased caspase 8 and 3 expression along with enhanced apoptosis. We also found the enhancement of apoptosis and Ca2+ concentrations had a positive correlation, which corresponds with the literature. In addition, the expression of both caspase 3 and 8 in the subcutaneous and preventive-injection groups showed some decrease, which may correlate with the low levels of Ca2+ concentration in the cells. This relationship needs further study.
The inhibitory effect of IFN-α-2b on tumor growth may be the result of three modes of action: 1)an indirect attack on the carcinoma cells by activating T and NK cells. 2) directly inhibiting carcinoma angiogenesis. 3) a direct inhibitory effect on the carcinoma cells. Our results indicate that IFN-α-2b takes part in an antitumoral effect by inducing cell apoptosis, corresponding to reports in the literature[15~17]. The apoptotic model we present can serve as an experimental basis for further research on the apoptotic mechanism in breast cancer cells. We realize that Ca2+, caspase-3 and 8 together play important roles in the apoptotic signaling transduction process of breast cancer. It is our hope that our research may develop into a new gene-therapeutic approach for breast cancer.
- Received December 21, 2006.
- Accepted April 20, 2007.
- Copyright © 2007 by Tianjin Medical University Cancer Institute & Hospital and Springer
In this issue
Jump to section
Related Articles
Cited By...
- No citing articles found.