| Lung cancer is one of the most common malignancies in the world and the most threatening cancer to human health. In China, lung cancer has become the leading cause of cancer mortality. Smoking is an important cause of tumorigenesis in lung; approximately,80%male and50%female patients have smoking history.’Tobacco control and China’s future’published by Center of Disease Control in China shows that smokers from2002to2010kept over300billion. To2030, the death caused by tobacco smoking will be more than3billion, accounting for25%of all death over40years. Therefore, prevention and treatment of smoking-induced lung cancer is the key point of current researches. Nicotine is the major addictive component in tobacco, which is able to induce tumor cell proliferation, promote angiogenesis and metastasis, and accelerate the progression of lung cancer by many means.There is no name for lung cancer in traditional Chinese medicine (TCM), but its symptom, cause and mechanisms have been described in ancient medical books. Ancient doctors considered that smoking is the cause of hot and hot is the mechanism why smoking results in lung cancer. Treatment of lung cancer by TCM has characteristics such as alleviate symptoms, improve life quality, prolong life time, moderate toxic effect made by chemical drugs and prevent metastasis. The main flavonoid components in Scutellaria baicalensis are baicalin, baicalein and wogonin that have wide anti-tumor effects. Previous studies show that flavonoid components in Scutellaria baicalensis inhibit the proliferation of many lung cancer cells; however, the effect of flavonoid components in Scutellaria baicalensis on nicotine-induced lung cancer progression has not been reported. Therefore, the present study focuses on the effect of flavonoid components in Scutellaria baicalensis on nicotine-induced lung cancer progression and the underlying mechanisms, which could provide an objective and scientific theoretical basis and experimental evidence for the anti-tumor effect of flavonoid components in Scutellaria baicalensis, offer effective drug and optimal scheme for clinical treatment of lung cancer, and open new avenue for lung cancer prevention. Here, our study is divided into three parts. Part1Effect of Flavonoid Components in Scutellaria Baicalensis on Nicotine-induced Proliferation and Apoptosis of Lung Cancer Cell Line A549and H1299Objective1. To investigate the biological effect of flavonoid components in Scutellaria baicalensis on lung cancer cell line A549and H1299;2. To investigate the mechanism of apoptosis of flavonoid components in Scutellaria baicalensis on lung cancer cell line A549and H1299.Methods1. In vitro, A549and H1299cells were exposed to nicotine (final concentration10"8M and10-6M respectively) and baicalin, baicalein and wogonin with different concentrations respectively (final concentration50μM,10μM andlμM). To observe the morphologic changes of the cells caused by nicotine itself and nicotine with baicalin, baicalein and wogonin. The effect of baicalin, baicalein and wogonin on nicotine-induced A549and H1299cells proliferation was detected by CCK-8, and effect on apoptosis was detected by flow cytometry;2. Effect of baicalin, baicalein and wogonin on nicotine-induced apoptosis-associated genes and proteins such as bcl-2, bax and pro-caspase-3in A549and H1299cells was detected by real-time PCR and western blot, in order to make the apoptosis mechanism clear.Results1. Flavonoid components in Scutellaria baicalensis significantly inhibited nicotine-induced A549and H1299cell proliferation, especially baicalein and wogonin. Intervened by flavonoid components in Scutellaria baicalensis, A549and H1299cells showed typical apoptosis behavior. Further, the result of Annexin V with PI staining method demonstrated the pro-apoptosis effect of flavonoid components in Scutellaria baicalensis. The early apoptosis rates of baicalin, baicalein and wogonin were6.90±0.40,19.63±1.23and22.5±3.22respectively in A549cell,11.82±2.73,14.04±4.27and12.55±2.74respectively in H1299cell;2. Intervened by flavonoid components in Scutellaria baicalensis on A549cell for 48h, baicalin, baicalein and wogonin significantly inhibited bcl-2mRNA and protein expression; baicalin significantly decreased bax mRNA expression, but had no effect on bax protein expression; wogonin significantly promoted bax mRNA expression, and baicalein and wogonin increased bax protein expression; baicalein and wogonin significantly inhibited bcl-2/bax mRNA and pro-caspase-3mRNA expression, and three components significantly declined bcl-2/bax and pro-caspase-3protein expression. Intervened by flavonoid components in Scutellaria baicalensis on H1299cell for48h, baicalin, baicalein and wogonin significantly inhibited bcl-2, bax and pro-caspase-3mRNA and protein expression; baicalein significantly decreased bcl-2/bax mRNA ratio, but had no effect on bcl-2/bax protein ratio.Part2Effect of Flavonoid Components in Scutellaria Baicalensis on Nicotine-induced Metastasis and Inflammatory Microenvironment of Lung Cancer Cell Line A549and H1299Objective1. To investigate effect of flavonoid components in Scutellaria baicalensis on nicotine-induced lung cancer metastasis;2. To investigate effect of flavonoid components in Scutellaria baicalensis on nicotine-induced lung cancer inflammatory microenvironment;3. To investigate mechanisms of flavonoid components in Scutellaria baicalensis on nicotine-induced lung cancer metastasis and inflammatory microenvironment.Methods1. Effect of baicalin, baicalein and wogonin (final concentration50μM,10μM and1μM) on nicotine-induced A549and H1299cells migration was detected by scratch study. Effect of baicalin, baicalein and wogonin on nicotine-induced A549and H1299cells metastasis was detected by transwell study;2. Effect of baicalin, baicalein and wogonin on nicotine-induced TNF-a and IL-6expression in A549and H1299cells was detected by ELISA;3. Effect of baicalin, baicalein and wogonin on nicotine-induced MMP-2, MMP-9, NF-κB p65and IκB-α mRNA and protein expression in A549and H1299cells was detected by real-time PCR and western blot respectively. Results1. Flavonoid components in Scutellaria baicalensis significantly inhibited nicotine-induced A549and H1299cell metastasis and migration, especially baicalein and wogonin. Baicalin, baicalein and wogonin significantly downregulated metastasis-associated MMP-2and MMP-9mRNA expression in A549and H1299cells; baicalein and wogonin significantly inhibited MMP-2and MMP-9protein expression in A549cell; three components significantly decreased MMP-2and MMP-9protein expression in H1299cell;2. Flavonoid components in Scutellaria baicalensis significantly inhibited nicotine-induced A549and H1299cell inflammatory microenvironment. Baicalin, baicalein and wogonin inhibited IL-6and TNF-a expression in A549cell and TNF-a expression in H1299cell; baicalin and baicalein decreased IL-6expression in H1299cell. Baicalin, baicalein and wogonin suppressed NF-κB p65mRNA and protein expression in A549and H1299cells, upregulated IκB-α mRNA and protein expression in H1299cell, but had no effect on IκB-α mRNA and protein expression in A549cell.Part3Effect of Flavonoid Components in Scutellaria Baicalensis on Nicotine-induced Lewis Lung Cancer in Mice and Its MechanismsObjectiveTo investigate the effect of flavonoid components in Scutellaria baicalensis on nicotine-induced Lewis lung cancer in mice and its mechanismsMethods1. A total of50mice were inoculated with Lewis lung cancer cells. One week after inoculation, the mice were randomly assigned to6groups,10mice in each group: control group, nicotine group, nicotine plus baicalin80mg/kg group, nicotine plus baicalein40mg/kg group and nicotine plus wogonin40mg/kg group. One week after grouping, except mice in control and nicotine groups were received intragastric gavage of sodium chloride, mice in other groups were received flavonoid components respectively once a day, a total of28days;2. Effect of baicalin, baicalein and wogonin on VEGF, TNF-a and IL-6expression of nicotine-induced Lewis lung cancer in mice was detected by ELISA;3. Effect of baicalin, baicalein and wogonin on bcl-2, bax and caspase-3expression of nicotine-induced Lewis lung cancer in mice was detected by western blot;4. Effect of baicalin, baicalein and wogonin on CD31and NF-κB p65expression of nicotine-induced Lewis lung cancer in mice was detected by immunohistochemistry, and microvascular density (MVD) was calculated by CD31expression.Results1. Flavonoid components in Scutellaria baicalensis significantly inhibited nicotine-induced Lewis lung cancer in mice, especially baicalein and wogonin. Baicalin, baicalein and wogonin suppressed nicotine-induced apoptosis-associated protein such as bcl-2and caspase-3expression, downregulated bcl-2/bax ratio; wogonin promoted nicotine-induced bax expression;2. Wogonin significantly inhibited nicotine-induced microangiogenesis of Lewis lung cancer in mice; baicalein had no effect on nicotine-induced microangiogenesis; baicalin significantly promoted nicotine-induced microangiogenesis of Lewis lung cancer in mice. All three flavonoid components in Scutellaria baicalensis significantly inhibited nicotine-induced VEGF expression of Lewis lung cancer in mice;3. Flavonoid components in Scutellaria baicalensis significantly inhibited nicotine-induced inflammatory microenvironment of Lewis lung cancer in mice. Baicalin, baicalein and wogonin suppressed IL-6expression, and baicalin and baicalein suppressed TNF-α expression. All three flavonoid components in Scutellaria baicalensis significantly inhibited nicotine-induced NF-κB expression of Lewis lung cancer in mice.Conclusion1. Flavonoid components in Scutellaria baicalensis significantly inhibited nicotine-induced proliferation in lung cancer cell lines A549and H1299, especially baicalein and wogonin. Flavonoid components in Scutellaria baicalensis dramatically suppressed nicotine-induced apoptosis-associated gene and protein such as bcl-2expression, upregulated bax expression, decreased bcl-2/bax ratio, downregulated pro-caspase-3expression, activated mitochondria apoptotic pathway in order to induce apoptosis;2. Flavonoid components in Scutellaria baicalensis significantly inhibited nicotine-induced migration and metastasis in lung cancer cell lines A549and H1299, especially baicalein and wogonin. The mechanism was possibly associated with down-regulation of metastasis-associated gene and protein such as MMP-2and MMP-9;3. Flavonoid components in Scutellaria baicalensis significantly inhibited nicotine-induced inflammatory microenvironment such as suppression of IL-6and TNF-a expression in lung cancer cell lines A549and H1299, which was possibly associated with the inhibition of NF-κB p65expression in A549and H1299and promotion of IκB-α expression in H1299;4. Flavonoid components in Scutellaria baicalensis significantly inhibited nicotine-induced Lewis lung cancer in mice, especially baicalein and wogonin. It is possibly related to downregulation of bcl-2and caspase-3expression and bcl-2/bax ratio. Effects of baicalin, baicalein and wogonin on nicotine-induced microangiogenesis of Lewis lung cancer were shown as promotion, no effect and inhibition; however, three flavonoid components in Scutellaria baicalensis significantly suppressed nicotine-induced VEGF expression of Lewis lung cancer in mice. In addition, flavonoid components in Scutellaria baicalensis significantly inhibited nicotine-induced inflammatory microenvironment of Lewis lung cancer in mice such as suppression of IL-6and TNF-a expression, which was possibly associated with the inhibition of NF-κB p65expression.
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