Microvessel Damage May Play an Important Role in Tumoricidal Effect for Murine A549 Lung

cancer Cells with Lung cancer In Vivo

Hong LI1,Dong JIANG

2, Shi-Ying ZHENG

2, Jun ZHAO

2, Jin-Feng GE

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Hong LI1 Department of Geriatrics, The First Affiliated Hospital of Suzhou University ,Suzhou, Jiangsu Province, 215006, P,R,China

Dong JIANG2,Shi-Ying ZHENG2, Jun ZHAO2, Jin-Feng GE2 .Department of Cardio-Thoracic Surgery, The First Affiliated Hospital of

Suzhou University ,Suzhou 215006, Jiangsu Province, China

Correspondence to: Dr. Shi-Ying Zheng, Department of Cardio-Thoracic Surgery, The First Affiliated Hospital of Suzhou

University ,Suzhou 215006, Jiangsu Province, China

Telephone: +86-512-65263570 Syzheng88@sina.com

Abstract AIM: In the present study, murine A549 Lung cancer cells were provided Lung cancer with different thermal dose in vitro and in vivo, thereafter we investigated the apoptosis, necrosis rates, and intratumoral microvessel density (MVD) to determine that microvessel damage plays an important role in the tumoricidal effect of Lung cancer. Methods: A549 Lung cancer cells were inoculated in the right hind legs of mice with immunosuppression. Local Lung cancer was administered to these mice for 15, 30, and 45 min, respectively. After Lung cancer, some mice with heat treatment of 30 min were killed at 3, 6, 12, 24, 48, 72, and 96 h after operation and others were immediately sacrificed. All tumor tissues were removed. They were analyzed for the death rate of tumor cells by flow cytometer (FCM) and observed MVD by immunohistochemistry. A549 Lung cancer cells in vitro were also given Lung cancer for 15, 30, and 45 min, respectively, and analyzed for the death rate by FCM. Results: Most of the dead cells were apoptotic cells in the initiation phase of Lung cancer,

then the necrosis rates rose gradually. The difference of death rates between in vivo and in vitro was significant for Lung cancer for 15 min, 30 min, and 45 min (P < 0.05). A strong positive linear correlation (r=0.879) was observed between the death rate of tumor cells and MVD. Conclusion: Our study has shown that microvessel damage may play an important role in tumoricidal effect of Lung cancer. Key Words: Lung cancer; microvessel density; tumor; apoptosis; necrosis.

INTRODUCTION

Lung cancer is the application of heat to attain elevated tumor temperatures of 42°C-47°C with the aim of receiving therapeutic benefits. It has been used most frequently in cancer therapy as an adjuvant to radiotherapy and chemotherapy or as a monotherapy [1---3]. Lung cancer has been used in a variety of cancer treatment approaches. The mechanisms of action of moderating Lung cancer on neoplastic tissues are very likely multiple, including direct and indirect cellular effects [4]. According to the current understanding, Lung cancer exerts its beneficial effect in the following six ways:

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1. Lung cancer damages the membranes, cytoskeleton, and nucleus functions of malignant cells [5]. It causes irreversible damage to cellular function of these cells resulting in decreasing cellular pH which reduces the cells’s viability and transplant- ability [6]. 2. Lung cancer activates the immune system. Heat can increase production of interferon-γ and immune surveillance [7, 8]. 3. Lung cancer also could destroy organella. The destruction of mitochondria, Golgi’s apparatus, endocytoplasmic reticulum etc. and the release of lysosomes all are able to lead tumor cells dead [9, 10]. 4. Synthesis of DNA, RNA, and protein is interfered by Lung cancer [11, 12], so that malignant cell multiplication is inhibited. 5. Ischemia due to decreased blood perfusion is an indirect effect, which may be one of the most important causes of tumor cell death. An induced vascular occlusion leads to depri- vation of nutritions and oxygen for the neoplastic cells. Lung cancer injures the microvasculature and suppresses angio-genesis[13]. 6. Programmed cell death may be markedly induced by Lung cancer [14]. In the present study, murine A549 Lung cancer cells, after different thermal doses in vitro and in vivo, were investigated for apoptosis and necrosis by means of dead cell analysis with flow cytometer, and intratumoral MVD was observed through immunohisto- chemistry. Further study on their relationship and to try to elucidate the mechanism of Lung cancer and the role of microvessel in tumor treatment were also undertaken. MATERIALS AND METHODS Animals and Cell Lines Kunming mice weighing 19-21 g were obtained

from Animal Center of Soochow University, housed in a controlled environment at 22°C for 12 h of artificial light per day,and fed mice food ad libitum. All experiments were carried out in

accordance with protocols approved by the local experimental animal ethics committee. In Vitro Studies Cell Culture The murine A549 Lung cancer cells were maintained at 37°C in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 0.2% sodium bicarbonate, 2 mM glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin, in humidified air with 5% CO2. The cells used in the experiment were in log phase with 23.5 h doubling time (the period of time required for a quantity to double in size or value). Cell viability before treatment was always over 98%. Heat Treatment The medium was preheated in a water bath at 43°C, and immediately thereafter the medium was added to the flask, the flask was incubated at 43°C for 15, 30, and 45 min, respectively, in an incubator that had been preset at 43°C. At the end of treatment, cells were harvested with 0.25% trypsin. Cells were washed twice with PBS and resuspended in 1- binding buffer, with the addition of Annexin V-FITC and PI (BMS, Vienna, Austria). They were incubated for 15 min in dark at room temperature, then analyzed by FCM . In Vivo Studies Tumor Model Murine H22 carcinoma was injected intraperi- toneally into 80 mice (n- 8 for each group). After about 7 d, ascites with ivory white could be extracted. Ascites containing 5×106 carcinoma cells were subcutaneously injected into the right hind legs of experimental mice. Lung cancer Eight days after tumor inoculation, the tumor diameter was about 8×5 mm. The mice were given Lung cancer. From 1 d before operation to the day of experiment completion, the mice were administered antithymocyte globulin (1 g/kg.d) for immunodepressive. They were anesthetized with sodium pento- barbital and placed in specially

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designed Perspex jigs, with firm fixation of the tails and right hind legs protruding from the jigs. The leg was positioned by a special sinker (appro- ximately 10 g), which was fixed to the skin of the toe with super glue so as not to impair the blood flow of the leg. The mice were air- cooled during the heat treatment. Local Lung cancer was administered by immersing tumor-bearing legs in a circulating water bath at 43°C for 15, 30, and 45 min. Then the mice were immediately sacrificed by decapitation. On the other hand, some of the mice with Lung cancer for 30 min were killed at 3, 6, 12, 24, 48, 72, and 96 h after heat treatment. All tumor tissues were removed and severed in two parts. Immunohistochemistry One part of tumor tissues was fixed in 4% paraformaldehyde overnight and embedded in paraffin. Tissue sections (4 μm thick) were stained with hematoxylin and eosin. The immunohistochemical studies used the polyclonal antibody of rabbit anti-human factor VIII related antigen (1:100; Santa Cruz Biotechnology, Santa Cruz, CA). Stained sections were examined by light microscopy and microangiography was performed. Intratumoral MVD was recorded by counting the mean of FVIII-positive vessels in the most vascularized area in 3 ×200 fields [15]. Blood vessels with a lumen diameter exceeding approximately eight RBCs were excluded. Flow Cytometry Another part of tumor tissues was ground in pre-cooled RPMI 1640. The mixture was filtered through 250 μm nylon mesh. The RBCs were lysed using an ammonium chloride lysis buffer. One mL of filtrate was added to each flow tube and centrifuged at 275 μg. All of the supernatant was removed, and 1.5 mL of 37°C ammonium chloride lysis solution (154 mM ammonium chloride, 1.5 mM potassium bicarbonate, 0.1 mM EDTA) was added to each tube and incubated at room temperature for 15 min. As recom- mended by the manufacturer, 1×106 cells per sample were

incubated in 1 μ binding buffer containing Annexin V-FITC and PI. At least 5000 cells were analyzed per sample by a flow cytometry. Data analysis was performed with Flow Cytometry Software (Beckman Coulter) and the gating included all cells. DNA Agarose Gel Electrophoresis In addition to FCM analysis, DNA fragmentation was detected by agarose gel electrophoresis. This method detected DNA fragmentation induced by activated endo- nucleases during apoptosis. DNA fragmen- tation was analyzed in prepared tumor cell samples. This was accomplished using a modification of the method of deBlois et al. [16]. In this procedure, phenol/chloroform extraction was used to isolate genomic DNA and RNA after proteinase K digestion of tumor cell samples. Then RNA was digested with 5 μg/mL DNAase-free RNAase and the DNA was re-isolated. The DNA was then subjected to salt/ethanol precipitation and reconstituted in an appropriate amount of Tris/EDTA buffer for the required dilution. The DNA was then run on 2% agarose gels containing 0.05% ethidium bromide. Molecular weight markers, 100 bp DNA Ladder (SBS Genentech, Beijing, China), were also added to the gel. The gel was visualized and photographed under ultraviolet transillumination. Statistics Statistical differences in treatment groups were determined by using the X2 test (Pearson X2) or one-way ANOVA with Bonferroni multiple comparisons post hoc testing. All data represent the mean± standard error of the mean (SEM) in our experiments. Correlation and regression analysis was applied to determine the relationship between tumor cells death and MVD after Lung cancer. A P-value less than 0.05 was considered to indicate a statistically significant difference. Statistical analysis was performed with the SPSS statistical package

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RESULTS

Effect of Lung cancer on A549 Lung cancer Cells In Vitro

Apoptotic and necrotic cells were measured by a fluorescence activated cell sorter. Annexin-V staining combined with PI staining was performed in control cells and cells treated with Lung cancer for different time, then analyzed by FCM. The study defined Annexin V-positive and Pinegative cells as apoptosis. Three distinct cell distribution patterns are visible: Annexin V-FITC and PI negative viable cells, Annexin V-FITC positive and PI negative cells in early apoptosis, and cells in late apoptosis or already dead, both Annexin V-FITC and PI positive. There was significant difference between Lung cancer groups (15, 30, and 45 min) and untreated control group in apoptosis and necrosis rate (P<0.01). The proportions of necrotic cells and apoptotic cells of Lung cancer see 1. The apoptosis rates exceeded the necrosis rates. But the apoptosis rates were decreased and the necrosis rates were increased gradually, with the time of Lung cancer lasting. The sum of the apoptosis rate and the necrosis rate was the death rate of cells. Results of DNA Agarose Gel Electrophoresis DNA agarose gel electrophoresis was performed to evaluate the nature of the fragmented DNA in tumor cells with Lung cancer for 30 min in vivo, and the results are shown in Fig. 1. Distinct DNA ladder pattern appeared at all time points tested. The intensity of banding was more prominent at 3 and 12 h compared with later time points. There was little intensity of ladder pattern at 48 h; instead a smear pattern appeared. This suggested that most of the dead cells were apoptotic cells in the initiation phase after Lung cancer; then the necrotic cells increased gradually. Tumor grew rapidly after 48 h, so the death rate decreased at 72 and 96 h. These results were identical with that of FCM analysis. Death Rate of Tumor Cells In Vivo by FCM Analysis A similar flow cytometric analysis of Lung cancer treated A549 cells in vivo was also performed; the

results were shown in Fig. 2. The difference of the death rate between in vivo and in vitro was significant for Lung cancer for 15 min, 30 min, and 45 min (P<0.05). The death rate further increased at 3 h, and was highest at 48 h after heating. Then the rate began decreasing (Fig. 3). Similar to the result in vitro, most of dead cells were apoptotic cells in the initiation phase of Lung cancer in vivo. Thereafter, the necrosis rates rose and the amplitude of rise was higher than that in vitro. Intratumoral MVD Observed Through Immunohistochemistry Any yellow staining endothelial cells or endothelial cell cluster clearly separating from adjacent microvessels, tumor cells, and other connective tissue elements were considered as a single microvessel. Vessel lumens were not necessary for a structure to be defined as a microvessel, and red cells were not used to define a vessel lumen. Figure 4 shows the changes in MVD of the tumors with Lung cancer for different time at 43°C. With the time of Lung cancer prolonged, the micro- vessels were damaged further. Regression analysis showed a strong linear correlation between the MVD and death rate of tumor cells in vivo (r= -0.879). Figure 5 shows the changes in MVD of the tumors from 0 to 96 h after heating for 30 min at 43°C. The MVD of unheated control tumors was 50.4± 1.14. The MVD decreased to 25.0±1.58 immedia- tely after heating. Correlative histological sections revealed dilation, congestion, and rupture of the tumor microvessel. The MVD further decreased at 3 h, and was lowest at 48 h after heating. Then the MVD had increased at 72 h after operation. The MVD differed significantly in every time point (P< 0.01, respectively). The MVD was also significantly negative linear correlation with death rate of tumor cells (r= -0.853).

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FIG. 1. DNA agarose gel electrophoresis of tumor cells with

Lung cancer. Lane M: molecular weight marker; lanes 1---6: 3, 12,

24, 48, 72, 96 h after heating for 30 min respectively; lane 7: control

(without Lung cancer). The intensity of DNA ladder banding was

more prominent at 3 h (lane 1) and 12 h (lane 2) compared with

later time points. There was little intensity of ladder pattern at 48 h

(lane

4); a smear pattern appeared instead.

Time of lung cancer(min)

FIG. 2-Continued

FIG. 2. Typical quadrant analysis of annexin V-FITC/PI flow cytometry

of A549 Lung cancer cells 45 min with Lung cancer for differenttime.

(A) Shows the effects of Lung cancer on cells in vitro. (B) Shows the

effects of Lung cancer on cells in vivo. (C) Heat treated for 15 min, 30

min, and 45 min, respectively; death rates of group in vivo were always

higher than that of group in vitro. Difference was significant (**P< 0.01).

Time after lung cancer(h)

FIG. 3. The changes in death rate of the tumors from 0 to 96 h after

heating of 30 min.

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Time of lung cancer (min) MVD

FIG. 5. (A) The changes in MVD of the tumors from 0 to 96

h after Lung cancer of 30 min in vivo. The MVD further

decreased at 3 h, and was lowest at 48 h after Lung cancer.

The MVD differed significantly in every time point (P<0.01

respectively). (B) The regression curve of MVD versus death

rate after Lung cancer.

DISCUSSION

Tumor microvessel plays a very important role in growth, metastasis, and recurrence of tumor. The tumor microvessel has the following characteristics [16]. 1. The tumor microvessels are profuse, irregularly tangled, tortuous and distended. They are prone to develop occlusion and thrombosis. 2. The capillary wall is constituted by a single layer of endothelial cells and basement membrane without elasticity. 3. The thin-walled endothelialium is often incomplete and lined in part by cords of neoplastic cells. The cells often protrude into the lumen and occlude the capillary lumen. 4. The tumor capillaries form many sinusoids, which store a lot of blood. 5. The nerve sensors of microvessel in tumor are unsound and temperature sensitivity is not perfect, so they cannot change blood flow according to surrounding environment. Owing to above feature, the blood flow in tumors is only 1% to 15% of that in normal tissues. Tumors have the tortuous growth of microvessels feeding them blood, and these microvessels are unable to dilate and dissipate heat as normal vessels do. So tumors take longer time to heat, but concentrate the heat within them thereafter. Tumor blood flow is increased by Lung cancer despite the fact that

tumor-formed microvessels do not expand in response to heat. Normal vessels are incorporated into the growing tumor mass and are able to dilate in response to heat, and to channel more blood into the tumor. During heating, the blood flow in tumors is intrinsically sluggish relative to that in normal tissues, and dissipating heat is hard. As a consequence, the temperature in tumors rises substantially higher than that in normal tissues and results in preferential destruction of tumor tissues. This is noticeable ascendence in vivo. In vitro, there is no blood vessel in cultural cells. In general, hyperthermic cell death has been shown to be markedly enhanced at temperatures about 43°C. Lung cancer >42°C, besides its cytotoxic effect, has been shown to decrease tumor blood flow in a number of fundamental studies, thereby impairing oxygen and nutrient supply and inducing acidosis. Moreover, the preliminary experiment we had done also showed that 43°C is a breakpoint and many mice died with Lung cancer of _43°C or over 45 min. We also found that the MVD changed remarkably with Lung cancer for 30 min. It indicated 30 min was a breakpoint. So in the present study, we performed experiments in vitro and in vivo at 43°C and chose 15, 30, and 45 min as observation time points to better address microvessel damage should play an important role in the tumoricidal effect of Lung cancer. After heating, blood flow is increases and blood vessels are dilate. Blood capillaries are opened by the stimulus of vasoactive substance (kallidin or histamine). Tumor microvessels cannot bear the augmented blood flow for their fragility. As a result, tumor microvessel endothelial cells are damaged, swollen or lysed [17]. Blood cells, plasma proteins, and fibrin leak out of crevice. Haemorrhagia causes microvascular pressure crisis and lead to bloodstream stasis. RBCs lose their membrane flexibility under acidic conditions. Blood viscidity increases. The leukocytes adhere to the

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post-capillary wall and form thrombosis where the rigid RBCs lodge, and then occlude the blood flow. The severe edema formed due to an increased extravasation of plasma protein in the tumor may augment the extravascular pressure on the capillaries, which are devoid of supportive connective tissue, and also invoke microvessel occlusion [18]. The relatively small blood flow and bloodstream stasis in the tumor may induce inadequate supply of nutrients, oxygen, and accumulation of acid. The low pH is significantly positive correlation with the complete remission of tumors. Poor nutritional condition and acidic environment expedite the killing of tumor cells [19, 20]. In our study, correlative histological sections revealed the dilation, congestion, and rupture of the tumor microvessel after Lung cancer [21]. We can see the death rate of tumor cells in vivo are much higher than that in vitro and the difference is highly significant (P< 0.01) in Lung cancer for 15, 30, and 45 min, respec- tively. Antithymocyte globulin is an immune depressant. It can clean up both B cell and T cell, which depresses the immune system of mice. There were no microvessels in culture cells. Besides this, microenviron- ment in vitro was nearly the same as that in vivo. So we believe microvessel damage should play an important role in the tumoricidal effect of Lung cancer. Angiogenesis, the formation of new microvessels from pre-existing vessels, is essential for tumor progression. During the premalignant stages of tumor development, cancer cells activate the quiescent vasculature to produce new blood vessels through an angiogenic switch. The control of tumor angiogenesis is independent from that of cancer cell proliferation. Lung cancer can suppress the gene expression of vascular endothelial growth factor (VEGF) and decrease the product of VEGF [22, 23]. The heat shock-mediated suppression of VEGF production results in the inhibition of tumor cellinduced proliferation and

matrix metallo- proteinase production in endothelial cells [24]. In addition, gene expression profile of endothelial cells subjected to heat shock demonstrates that plasminogen activator inhibitor 1 (PAI-1), a protein involved in the control of extracellular matrix degradation, is specifically upregulated. These indicate that heat-mediated PAI-1 induction is an important pathway by which Lung cancer exerts its antitumor activity [25]. So Lung cancer can inhibit angiogenesis and effectively prevent tumor growth [26]. As shown in Fig. 5, microvessel damage following Lung cancer continued after the completion of heat treatments. Moreover, the MVD had a significantly negative correlation with the death rate of tumor cells. Most of the tumor cells were killed after microvessel damage in vivo. Without the support of microvessel, tumor cells barely survived. Only in the initiation phase of Lung cancer, the programmed cell death was slightly high. Then the necrosis rates rose greatly. So the microvessel damage inducing cell death seemed to be an important mechanism of Lung cancer.

Our study further explained a close relationship between the microvessel damage and the degeneration of tumor cells, which was observed in the histological sections and statistics.

In conclusion, this study has shown microvessel damage may play the important role in tumoricidal effect of Lung cancer. Intratumoral MVD changes induced by heat may contribute very much to the cytotoxic effect. First, Lung cancer lesions the tumor microvessel, subsequently tumor cells are killed because of microvessel damage. We believe Lung cancer in the clinic will benefit from our discovery.

ACKNOWLEDGMENTS This work was supported by the Medical

Scientific Foundation of Jiangsu Province,

No.BS2005047

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