Application of matriculene in preparation of medicine for treating non-small cell lung cancer

Abstract

The invention discloses an application of matriculene in preparation of a medicine for treating non-small cell lung cancer. According to the application disclosed by the invention, matriculene inhibits proliferation of non-small cell lung cancer cells in a G1 phase by arresting a cell cycle, induces apoptosis through a mitochondrial pathway and inhibits migration and invasion of the cells, and growth of transplanted tumors is inhibited by inducing apoptosis of transplanted tumor cells in a nude mouse transplanted tumor model, so that an anti-tumor biological effect is exerted; the compound has the prospect of research and development of new anti-tumor drugs for treating non-small cell lung cancer and the like.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and particularly relates to application of matricaria chamazulene in preparing a medicine for treating non-small cell lung cancer. Background Art

[0002] According to global cancer statistics in 2020, lung cancer is the most common cancer, accounting for 11.4% of the total cancer incidence and 18.0% of the total cancer deaths (CA Cancer J Clin, 2021, 71: 209-249). Moreover, non-small cell lung cancer is one of the leading causes of cancer deaths worldwide (Cancer Discov, 2022, 12: 1676-1689). Currently, chemotherapy combined with surgery and radiotherapy has been used to treat lung cancer, but its side effects, such as nephrotoxicity and neurotoxicity, are detrimental to patient survival (Mol Cell Biochem, 2021, 476 (1): 57-68). Therefore, there is an urgent need to develop effective drugs with less toxic side effects. Natural products are an important source of anti-tumor drugs. Elemene derived from volatile oil of Curcuma zedoaria has been developed into anti-tumor drugs: elemene emulsion injection and oral emulsion, of which elemene emulsion injection has been used to treat lung cancer.

[0003] Matricariae chamomillae (also known as chamomile ketone, CAS: 000529-05-5) is a rare blue oily substance. Its color comes from the conjugated double bond system in the molecule (such as the cyclic terpene structure). This characteristic is extremely rare in natural products. Its molecular formula (C 14 H 16 ) and low melting point (<25°C) also make it unique among volatile oil components, and it is mainly found in the Chinese herbal medicine Matricaria chamomilla L. (Horticulturae, 2022, 8(12):1135). Matricaria chamomilla has great development value in medicine, but there are few studies on the pharmacological activity of Matricaria chamomilla, and no reports on its anticancer effect have been found. Summary of the invention

[0004] The purpose of the present invention is to provide the use of matricaria in the preparation of a drug for treating non-small cell lung cancer, to explore new uses of matricaria, and to provide a new choice for drugs for treating non-small cell lung cancer.

[0005] The present invention also found that Matricaria recutita inhibits the proliferation of non-small cell lung cancer through G1 phase arrest, induces apoptosis through mitochondrial pathway, inhibits the metastasis of non-small cell lung cancer cells, and inhibits the growth of transplanted tumors in vivo.

[0006] The technical solution adopted by the present invention is as follows:

[0007] Through experimental research, the present invention discovers that chamazulene has better cytotoxic activity against human non-small cell lung cancer cells than the positive control elemene emulsion injection, and at the same time has lower cytotoxicity to normal cells, showing low toxicity and high anti-tumor activity; it has an obvious inhibitory effect on the proliferation of human non-small cell lung cancer cells through G1 phase arrest; it promotes the apoptosis of human non-small cell lung cancer cells through the mitochondrial apoptosis pathway; it has a significant inhibitory effect on the metastasis of human non-small cell lung cancer; and it inhibits the growth of nude mouse xenografts by inducing apoptosis of cancer cells.

[0008] By adopting the above technical solution, the present invention discovers for the first time a new use of chamazulene in the treatment of non-small cell lung cancer, provides a new option for drugs for treating human non-small cell lung cancer, and has important application value in the pharmaceutical industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 It is a comparison of the cytotoxicity of chamazulene against non-small cell lung cancer cells (A549), human prostate cancer cells (PC-3), non-small cell lung cancer cells (H1299) and normal human embryonic lung fibroblasts (MRC-5);

[0010] Figure 2 It is the effect of chamazulene on the colony formation of A549 cells;

[0011] Figure 3 It is the effect of chamazulene on the cell cycle of A549 cells. (A) After treatment with chamazulene, the detection result of the cell cycle of A549 cells by flow cytometry, (B) The statistical result of the proportion of A549 cells in different periods (G1, S, G2);

[0012] Figure 4 It is the effect of chamazulene on the levels of cell cycle-related proteins in A549 cells. (A) After treatment with chamazulene, the Western blot result of cell cycle-related proteins, (B) The quantitative statistical result of the gray value of the Western blot bands;

[0013] Figure 5 It is the morphological observation of the apoptosis of A549 cells induced by chamazulene. (A) The morphological effect of chamazulene on A549 cells, (B) AO / EB staining analysis of the apoptosis of A549 cells induced by chamazulene, (C) Hoechst 33258 staining analysis of the apoptosis of A549 cells induced by chamazulene;

[0014] Figure 6 It is the analysis of the apoptosis of A549 cells induced by chamazulene by flow cytometry. (A) After A549 cells are treated with different concentrations of chamazulene for 24 h, stained with Annexin V-APC / PE, and analyzed by flow cytometry, (B) The statistical result of the proportion of total apoptotic cells and live cells;

[0015] Figure 7 To analyze the effect of chamazulene on the mitochondrial membrane potential of A549 cells by JC-1 staining;

[0016] Figure 8 To study the effect of chamazulene on the proteins related to the mitochondrial apoptosis pathway in A549 cells. (A, C) After treatment with chamazulene for 48 h, the expression levels of total Cyt c, Bax, Bcl-2, pro-caspase9, pro-caspase 3, and cleaved-PARP proteins related to apoptosis, (B, D, E) Quantitative statistical results of the gray values of the bands of total Cyt c, Bax, Bcl-2, pro-caspase 9, pro-caspase 3, and cleaved-PARP;

[0017] Figure 9 To investigate the effect of chamazulene on the migration, invasion, and expression of related proteins in A549 cells. (A, B) The migration ability of A549 cells treated with chamazulene was detected by wound healing assay and quantitatively analyzed by the migration rate (%). (C, D) The invasion ability was determined by transwell invasion assay (×100) and quantitatively analyzed. (E, F) WB was used to detect the expression of metastasis-related proteins (MMP-2 and N-cadherin) in A549 cells after treatment with chamazulene and the quantitative statistical results of the gray values of the bands of MMP-2 and N-cadherin.

[0018] Figure 10 To study the in vivo anti-tumor effect of chamazulene in a nude mouse xenograft model. (A) The effect of chamazulene on the volume curve of transplanted tumors. (B) Representative pictures of transplanted tumors in each group 15 days after administration. (C) The effect of chamazulene on the weight of transplanted tumors. (D) The inhibition rate of chamazulene on transplanted tumors.

[0019] Figure 11 For HE staining and TUNEL staining of transplanted tumors (×200). (A) HE staining was used to observe the tissue morphology of nude mouse transplanted tumors. (B) TUNEL staining was used to detect apoptosis of transplanted tumor cells. Specific implementation methods

[0020] Pharmacological example 1: Cytotoxicity of chamazulene against human non-small cell lung cancer cells (A549), human prostate cancer cells (PC-3), human non-small cell lung cancer cells (H1299), and normal human embryonic lung fibroblasts (MRC-5)

[0021] All experiments were carried out using complete medium, namely RPMI-1640 medium containing 10% fetal bovine serum, 2 mM glutamine, 100 U / mL penicillin and 100 μg / mL streptomycin. Cells in the logarithmic phase were selected, digested with trypsin, counted using a hemocytometer, and then diluted to the required cell concentration with RPMI-1640 complete medium; 80 μL was inoculated into a 96-well plate (5×10 3 cells per well), and cultured in a CO2 cell incubator for 24 h; after the cells were completely stable or adhered, 20 μL of the chamazulene medicinal solution diluted with RPMI-1640 medium was added to each well. The negative control group was an equal volume of medium. Each group had 5 parallel wells and was continued to be cultured in a 37 °C, 5% CO2 incubator for 48 h; after co-culture, 10 μL of MTT (5 mg / mL) was added to each well, and then incubated in a 37 °C, 5% CO2 incubator for 4 h; the 96-well plate was taken out, and the culture medium supernatant was aspirated. 150 μL of DMSO was added and shaken for 10 min to completely dissolve the product of the MTT reaction with DMSO. The OD value at a wavelength of 490 nm was read using an enzyme-linked immunosorbent assay reader. The results were repeated three times and the average value was taken.

[0022] The calculation formula for the inhibition rate is as follows: cell line inhibition rate = [1 - (OD value of the sample group - OD value of the blank control well) / (OD value of the control group - OD value of the blank control well)] × 100%.

[0023] Using SPSS 25.0 analysis software, calculate the concentration of the sample required to inhibit the proliferation of half of the cells (IC 50 ), and use IC 50 to evaluate the cytotoxicity of the sample. The results are shown in Table 1. Chamazulene has the best cytotoxic activity against human non-small cell lung cancer cell line A549 (IC 50 = 40.16 ± 0.22 μg / mL), and the effect is better than that of the positive control elemene emulsion injection (IC 50 = 45.98 ± 2.09), and at the same time, the cytotoxicity of MRC-5 (IC 50 = 91.14 ± 1.80 μg / mL) is relatively low. In addition, it shows good cytotoxic effects on human tumor cell line PC-3 (IC 50 = 62.52 ± 1.87 μg / mL) and human non-small cell lung cancer cell line (H1299) (IC 50 = 94.64 ± 8.46 μg / mL).

[0024] By comparing the inhibitory effects of chamazulene on A549, PC-3, H1299, and MRC-5 (as Figure 1As shown, chamazulene has a better effect on A549 cells than the positive control drug elemene emulsion injection, and has lower toxicity to normal cells, indicating that chamazulene has good selectivity for cancer cells and has the characteristics of low toxicity and high anti-tumor activity.

[0025] Table 1 Cytotoxic activity of chamazulene on tumor cells and normal cells

[0026]

[0027] Pharmacological Example 2: Effect of chamazulene on the proliferation of A549 cells (plate colony formation assay)

[0028] Take A549 cells in the logarithmic phase. After digestion with 0.25% trypsin, resuspend them with RPMI-1640 (containing 10% fetal bovine serum) culture medium. After counting with a hemocytometer, inoculate 200 cells per well into a 6-well plate, and pipette to disperse the cells. Place them in a CO2 incubator and culture for 24 h. Discard the culture medium. According to the blank group and the chamazulene treatment group, administer 0, 15, 20, and 25 μg / mL respectively. Set three replicates for each group and continue routine culture. After 3 d, replace the fresh culture medium and continue to culture for about one week. Observe under an inverted microscope at any time to check the cell proliferation status and colony formation. Until cell colonies visible to the naked eye are formed. Aspirate the culture medium, wash twice with PBS, add 800 μL of fixative to each well, let it stand at room temperature for 30 min, then discard the fixative, and add 800 μL of 0.1% crystal violet staining solution for staining. After placing it in the dark for 15 min, discard the staining solution, wash off the staining solution with distilled water at the bottom, and let it stand at room temperature to evaporate the distilled water. Place the 6-well plate under a white background cloth, take pictures and count, and calculate the monoclonal rate.

[0029]

[0030] Through the plate colony formation assay, the effect of chamazulene on the colony formation of A549 cells was investigated to explore its inhibitory effect on the proliferation of A549 cells. The results are as follows Figure 2 shown. Compared with the blank group without chamazulene treatment, chamazulene significantly reduced the number and size of A549 cell colonies ( Figure 2 A). As shown in Figure 2 B, the colony formation rates of chamazulene (15, 20, and 25 μg / mL) were 44.83 ± 2.72%, 21.17 ± 2.49%, and 1.33 ± 0.62% respectively, which were significantly lower than those of the blank control group (51.33 ± 3.06%) (p < 0.05 or p < 0.001), indicating that chamazulene significantly reduced the clone formation rate of A549 cells in a dose-dependent manner. In summary, chamazulene significantly reduced the number and size of colonies, and significantly reduced the clone formation rate of A549 cells in a dose-dependent manner, and had a significant inhibitory effect on the proliferation of A549 cells.

[0031] Pharmacological Example 3: Effects of Matricaria chamomilla on the cell cycle of A549 cells

[0032] A549 cells in logarithmic phase were taken and counted using a hemacytometer. 5 The cells were inoculated into 6-well plates and placed in a CO2 incubator for 24 h. The culture medium was discarded, and the blank group and the Matricariae chamomile treatment group were given 0, 20, 40, 80, 100 and 160 μg / mL of drugs, respectively. Three replicates were set in each group, and the conventional culture was continued for 24 h. The cells were digested by trypsin, collected in EP tubes, centrifuged at 800 r / min for 10 min, the culture medium was discarded, and the cells were rinsed with PBS. After centrifugation and discarding the supernatant, the cells were resuspended with PBS, and the cell concentration was counted and adjusted to 1×10 6 / mL; take 1mL of cell suspension into an EP tube, centrifuge and discard the supernatant, rinse with PBS, centrifuge and discard the supernatant; add Permeabilization solution (10μL) and DNAStaining solution (1mL), vortex for 5-10 seconds to mix, incubate at room temperature in the dark for 30min; detect the red fluorescence at an excitation wavelength of 488nm on a flow cytometer, and then analyze the various phases of the cell cycle.

[0033] The effect of Matricaria chamomilla on the cell cycle of A549 cells was detected by flow cytometry. Figure 3 As shown. Compared with the blank group without Matricaria zinnia treatment, Matricaria zinnia significantly increased the proportion of G1 phase cells, and significantly downregulated the proportion of S phase and G2 phase cells, resulting in G1 phase arrest. Compared with the control group (48.63±0.76%), the proportion of cells in the G1 phase of A549 cells (20, 40, 80, 100 and 160 μg / mL) was significantly increased, which was 50.63±1.07%, 50.81±1.13%, 56.62±0.70%, 57.21±1.45% and 61.69±0.76%, respectively. It shows that Matricaria zinnia significantly increased the proportion of G1 phase cells in a dose-dependent manner (p<0.001). Therefore, Matricaria zinnia significantly increased the proportion of G1 phase cells, resulting in G1 phase arrest of A549 cells.

[0034] Western blot was used to study the effect of Matricaria chamomilla on the expression of A549 cell cycle-related proteins. Figure 4As shown, compared with the untreated blank group, the expression level of p21 increased with the increase in the concentration of chamazulene (p < 0.01 or p < 0.001). In addition, after treatment with chamazulene, the protein expression levels of Cyclin E2, CDK2, CDK4, and Cyclin D3 decreased significantly in a concentration-dependent manner (p < 0.05 or p < 0.01 or p < 0.001). The results showed that chamazulene triggered cell cycle arrest of A549 cells in the G1 phase by reducing the Cyclin E2-CDK2 and Cyclin D3-CDK4 complexes and increasing p21, and hindered the transition from the G1 phase to the S phase. Pharmacological Example 4: Effect of chamazulene on apoptosis of A549 cells

[0035] (1) Administration treatment: Take A549 cells in the logarithmic phase. After digestion with 0.25% trypsin, resuspend them with RPMI-1640 (containing 10% fetal bovine serum) culture medium; after counting with a hemocytometer, inoculate 5×10 5 cells / well into a 6-well plate and culture them in a CO2 incubator for 24 h; discard the culture medium, add 2 mL of drug-containing culture medium (0, 20, 40, 80, 100, and 160 μg / mL), and continue to culture for 48 h.

[0036] (2) After the administration treatment, observe the morphological changes of A549 cells under an inverted microscope.

[0037] (3) After the administration treatment as in (1), perform AO / EB staining. Mix the AO staining solution and the EB staining solution in a 1:1 ratio and vortex to dissolve them thoroughly; discard the culture medium, gently wash once with pre-cooled PBS buffer, add 800 μL of AO / EB staining solution to each well, incubate in the dark for 5 min; aspirate the staining solution, observe under an inverted fluorescence microscope, take pictures and analyze.

[0038] (4) After the administration treatment as in (1), perform Hoechst 33258 staining. Discard the culture solution, add 0.5 mL of fixing solution to each well, fix for 10 min, discard the fixing solution, wash twice with pre-cooled PBS, shake back and forth several times, and aspirate the liquid; add 0.5 mL of Hoechst 33258 staining solution to each well, stain for 5 min, shake several times during the period, discard the staining solution, wash twice with pre-cooled PBS, shake back and forth several times, and aspirate the liquid; observe under an inverted fluorescence microscope, take pictures and analyze.

[0039] As Figure 5 shown in A, after treating A549 cells with different concentrations of chamazulene for 48 h, with the increase in the concentration of chamazulene, the number of shrunken cells increased significantly; as shown in AO / EB Figure 5 in B, after treatment with different concentrations of chamazulene, the proportion of green fluorescence gradually decreased, while the proportion of orange-red fluorescence gradually increased, indicating that the number of apoptotic cells gradually increased; Hoechst 33258 asFigure 5 As shown in C, after treatment with different concentrations of chamazulene, the proportion of cells showing bright blue fluorescence with dense nuclei gradually increased, showing the characteristics of apoptosis. All of the above morphological observations indicated that chamazulene could effectively induce apoptosis of A549 cells.

[0040] Annexin V-APC / PE staining combined with flow cytometry was used to further quantitatively detect the apoptosis-inducing effect of chamazulene on A549 cells. The results are as Figure 6 shown. Compared with the blank group (apoptosis rate: 0.73±0.38%), after A549 cells were treated with 20 μg / mL, 40 μg / mL, 80 μg / mL, 100 μg / mL, and 160 μg / mL chamazulene for 24 h, the proportions of apoptotic A549 cells increased to 7.94±0.60%, 17.08±1.04%, 36.22±2.10%, 43.64±0.51%, and 54.53±1.59%, respectively, indicating that chamazulene significantly increased the proportion of apoptosis in a dose-dependent manner (p<0.001), and at the same time chamazulene significantly decreased the proportion of live cells. Therefore, chamazulene significantly induced apoptosis of A549 cells in a dose-dependent manner.

[0041] The downregulation of mitochondrial membrane potential is a hallmark event of apoptosis. JC-1 is a fluorescent probe for detecting mitochondrial membrane potential (ΔΨm). When the mitochondrial membrane potential is high, JC-1 forms polymers in the mitochondrial matrix, producing red fluorescence; when the mitochondrial membrane potential is low, JC-1 cannot form polymers in the mitochondrial matrix, and JC-1 is a monomer, producing green fluorescence. The ΔΨm value of mitochondria is measured by red and green fluorescence. The results of JC-1 staining are as Figure 7 shown. After A549 cells were treated with different concentrations of chamazulene for 48 h, the proportion of red-fluorescent cells gradually decreased, and the proportion of green-fluorescent cells gradually increased,. The results showed that chamazulene could effectively cause the downregulation of mitochondrial membrane potential (ΔΨm) in A549 cells, thus leading to apoptosis.

[0042] The expression of proteins related to the mitochondrial apoptosis pathway was studied by WB experiment. As Figure 8 shown in A and B. Compared with the untreated blank group, chamazulene at concentrations of 40, 60, and 80 μg / mL could significantly upregulate the level of Cyt c in total protein (p<0.05 or p<0.001), suggesting that chamazulene inhibited the release of mitochondrial Cyt c.

[0043] From Figure 8 C and D, it can be seen that with the increase of chamazulene concentration, the ratio of Bax / Bcl-2 also increased significantly (p<0.01 or p<0.001). At Figure 8In C and E, chamazulene significantly decreased the protein levels of Pro-Caspase 3 and Pro-Caspase 9 (p<0.05 or p<0.01 or p<0.001), while upregulating the protein level of Cleaved-PARP (p<0.001). Therefore, chamazulene induced apoptosis of A549 cells through the mitochondrial apoptosis pathway by increasing the Bax / Bcl-2 ratio, leading to the release of Cyt c, which in turn activated the caspase 9 and caspase 3 cascade reactions, resulting in the cleavage of the apoptosis marker protein PARP. Pharmacological Example 5: Effect of chamazulene on the metastasis of A549 cells

[0044] Scratch: Collect the A549 cell suspension and dilute it to 2×10 5 cells / mL, then add 4×10 5 cells to a six-well plate. Culture overnight until the cells cover the bottom of the six-well plate to form a monolayer of confluent cells. Subsequently, use the tip of a 200 μL pipette to draw a straight line on the monolayer cells in each well. Next, wash away the cells floating due to scratching with PBS, and then add different concentrations of chamazulene (prepared with serum-free medium) and treat for 48 h. The scratched area is photographed with a Leica microscope at 0 h and 48 h. The cell migration rate is calculated as follows:

[0045]

[0046] Invasion: Add 750 μL of drug solutions with different concentrations (15% FBS) to the lower chamber. Inoculate 250 μL of A549 cell suspension (1×10 5 cells / well) into the upper chamber and add 250 μL of drug solutions with different concentrations (containing 5% FBS). After 48 h of incubation, fix with 10% paraformaldehyde for 2 minutes; incubate with absolute methanol for 20 minutes; stain with 0.1% crystal violet for 15 minutes; wash twice with PBS and then take images with a Leica DMi8 microscope. Use ImageJ software to quantify the number of invasive cells in each field of view and perform statistical analysis.

[0047] The wound healing assay and invasion assay were used to study the effect of chamazulene on the migration and invasion abilities of A549 cells. As Figure 9 shown in A and B, compared with the blank control group (88.23±0.91%), the lateral migration ability of A549 cells treated with different concentrations of chamazulene (10, 20, 30, and 40 μg / mL) gradually decreased, and the migration rates were 61.62±0.67%, 44.87±1.33%, 34.14±1.77%, and 24.43±1.28% respectively (p<0.001). Therefore, the inhibition of the migration ability of A549 cells by chamazulene was significantly concentration-dependent. The results of the invasion assay( Figure 9C and D) showed that compared with the control group, matricaria azulene could significantly reduce the number of invading A549 cells, and it was dose-related, indicating that matricaria azulene could inhibit the invasion ability of A549 cells.

[0048] The effect of matricaria azulene on metastasis-related proteins was detected by WB. As Figure 9 shown in E and F, the expression level of N-cadherin decreased with the increase of matricaria azulene dose (p<0.01 or p<0.001). In addition, the expression levels of MMP-2 were significantly down-regulated at 60 μg / mL and 80 μg / mL (p<0.01 or p<0.001). The above results indicated that matricaria azulene inhibited the migration and invasion abilities of A549 cells by down-regulating the levels of MMP-2 and N-cadherin.

[0049] Pharmacological Example 6: Anti-lung cancer effect of matricaria azulene in vivo

[0050] Furthermore, a nude mouse xenograft model was used to explore the anti-tumor effect of Achillea wilsoniana volatile oil in vivo. Cells (2×10 7 cells / mL, 200 μL) were inoculated into the left anterior limb axillary region of BALB / c-nu nude mice. When the volume (V = 0.52×length×width2) ≥ 100 mm 3 , the model was determined to be successfully constructed. After the tumor model was successfully constructed, the experimental nude mice were randomly divided into three groups, with 10 mice in each group. The specific grouping was as follows: the model group, that is, the solvent control group, only received the basic solvent treatment, and the solvent system consisted of normal saline, Tween 80 (final concentration 0.3%) and DMSO (final concentration 0.03%); the positive control group received elemene emulsion injection intervention (100 mg / kg); the Achillea wilsoniana volatile oil group received Achillea wilsoniana volatile oil (100 mg / kg, dissolved in normal saline, 0.3% Tween 80, 0.03% DMSO solution). Each group of nude mice was administered once every 48 h for a total of 15 days.

[0051] As Figure 10 shown in A and B, the tumor volume of the model group increased at a relatively fast rate over time, while the matricaria azulene group (100 mg / kg) and the positive control group (elemene, 100 mg / kg) showed a slow growth trend, and the growth rate of the matricaria azulene group was significantly slower than that of the elemene group. After 15 days of administration, compared with the model group (1931.21±202.61 mm 3 ), both the matricaria azulene group (100 mg / kg) and the positive control elemene group (100 mg / kg) could significantly inhibit the growth of transplanted tumors, and the volumes of transplanted tumors were 326.53±61.91 mm 3 and 444.77±50.06 mm 3 (p<0.001). It should be noted that the tumor volume of the matricaria azulene group was significantly smaller than that of the elemene group. AsFigure 10 As shown in Figures C and D, compared with the model group (1.95 ± 0.10 g), the tumor masses in the chamazulene group (100 mg / kg) and the positive control elemene group (100 mg / kg) were significantly reduced, being 0.66 ± 0.07 g and 0.72 ± 0.08 g respectively (p < 0.001). In addition, the tumor mass in the chamazulene group was comparable to that in the elemene group (p > 0.05). Meanwhile, the tumor inhibition rate in the chamazulene group (65.96% ± 3.52%) was comparable to that in the elemene group (65.81% ± 5.38%) (p > 0.05). The above results indicate that chamazulene has an inhibitory effect on the growth of transplanted tumors in nude mice.

[0052] The HE staining results of the transplanted tumor tissues are as Figure 11 shown in Figure A. In the model group, the tumor cells had a high nuclear-cytoplasmic ratio, large volume, were closely arranged, and had uniform morphological sizes. Compared with the model group, in the transplanted tumor tissues of the chamazulene group (100 mg / kg) and the elemene group (100 mg / kg), the number of cells with shrunken cells and condensed nuclei increased significantly, and the cells were loosely arranged with larger cell gaps. This indicates that chamazulene can induce apoptosis of A549 transplanted tumor cells, thereby inhibiting the growth of transplanted tumors in nude mice. The TUNEL staining results are as Figure 11 shown in Figure B. In the model group, the tumor cells grew vigorously and there were very few apoptotic cells, almost completely showing blue fluorescence. Compared with the model group, the proportion of apoptotic cells showing red fluorescence in the chamazulene group (100 mg / kg) and the elemene group (100 mg / kg) increased significantly. The above results indicate that chamazulene can induce apoptosis of tumor cells, thereby inhibiting the growth of transplanted tumors in nude mice.

[0053] Based on the above pharmacological examples, chamazulene inhibits proliferation by triggering cell cycle arrest of A549 cells at the G1 phase through Cyclin E2-CDK2↓, Cyclin D3-CDK4 complex↓, and p21↑; the molecular mechanism of inducing apoptosis through the mitochondrial-mediated pathway is as follows: Bax / Bcl-2↑ → ΔΨm↓ → Total Cyt C↑ leads to the release of Cyt C from mitochondria → activation of Caspase-9 → activation of Caspase-3 → inactivation of PARP → induction of apoptosis; the inhibition of the migration and invasion of A549 cells is achieved through MMP-2↓ and N-cadherin↓; in the nude mouse transplanted tumor model, the growth of transplanted tumors is inhibited by inducing apoptosis of transplanted tumor cells.

Claims

1. Use of chamazulene in the preparation of a drug for treating non-small cell lung cancer.

2. The application according to claim 1, wherein The drug for treating non-small cell lung cancer is a drug that causes G1 phase arrest, thereby inhibiting the proliferation of non-small cell lung cancer cells.

3. The application according to claim 1, characterized in that The drug for treating non-small cell lung cancer is a drug that induces apoptosis of non-small cell lung cancer cells through the mitochondrial apoptosis pathway.

4. The application according to claim 1, characterized in that, The drug for treating non-small cell lung cancer is a drug that inhibits the metastasis of non-small cell lung cancer cells.

5. The application according to claim 1, characterized in that, The drug for treating non-small cell lung cancer is a drug that inhibits the growth of transplanted tumors by inducing apoptosis in a nude mouse transplanted tumor model.

6. Use of chamazulene according to claim 1 in the preparation of a medicament for treating non-small cell lung cancer, characterized in that, Chamazulene and a pharmaceutically acceptable carrier are made into tablets, capsules, fat emulsions, suppositories, dripping pills or ointment dosage forms of drugs.

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