br It is common that lung cancer
It is common that lung cancer patients have metastasis, which leads to treatment failures [4,16,17]. Metastatic lung cancer is characterized by uncontrolled cell growth in tissues of the lung, which can spread beyond the lung into nearby tissues or other parts of the body by the process of metastasis . To evaluate the inhibition ability of CAA45 on the migration of A549 cells, we performed wound healing and transwell assays. Our data demonstrated that CAA45 significantly in-hibited A549 Prostaglandin J2 migration by reducing MMP-2 and MMP-9 expres-sions, which might lead to inhibition of cell proliferation.
CAA was reported to induce cell apoptosis , but their mediated signaling pathways remained unknown. In our study, we found that CAA analogue CAA45 induced A549 cell apoptosis in a concentration-dependent manner. Our results provided more detailed information on the molecular mechanisms by which CAA45 induces apoptosis in A549 cells (i.e. by enhanced cytochrome c release, caspase-3, -8 and -9 cleavage activation, increased expression of Bax and Bad, and
Fig. 11. CAA45 significantly inhibited tumor growth in A549 mouse xenograft model. Male BALB/c nude mice (5–7 weeks of age) were inoculated with A549 cells. After tumor inoculation, the mice were randomly assigned into 4 treatment groups (3 mice/group) of vehicle, CPT (5 mg/kg), CAA45 (5 mg/kg) and CAA45 (10 mg/ kg) was administered by intramuscular injection twice a week. (A) Average tumor volume and (B) body weight changes of mice during 6 weeks of exposure. Error bars represent means ± SEM for a total of 3 samples. After the 42-day drug administration, tumor tissues of each samples were harvested, photographed (C), sectioned and subjected to H&E staining (D) for evaluating histological morphology under fluorescent microscopy (40×).
decreased expression of Bcl-2), which is clearly related to the mi-tochondrial mediated apoptosis pathway [20,21]. Up-regulated Bad and Bax and down-regulated Bcl-2 expressions led to the release of cytochrome c from mitochondria to the cytoplasm, subsequently acti-vating the caspase-3, caspase-8 and caspase-9 signal pathways. Caspase-3 activation is recognized as a biomarker for cells undergoing apoptosis [22,23]. Therefore, CAA45 induced apoptosis occurs via a mitochon-drial-mediated pathway in A549 cells.
Apoptosis and autophagy have coordinated and cooperative inter-actions to promote cell death under certain circumstances, and type I apoptosis and type II autophagic cell death collaborate to kill cells [24,25]. Our study has shown that CAA45 not only induced apoptosis but also enhanced autophagy in A549 cells, suggesting that inhibition of lung cancer cell proliferation by CAA45 was promoted by both au-tophagy and apoptosis. Furthermore, treatment of cells with CAA45 resulted in inactivation of JNK and Akt, and up-regulation of p53. It has been reported that the PI3K/Akt pathway, MAPK (JNK) signaling and p53 play important roles in cell proliferation, apoptosis and autophagy [26–29]. It is possible that CAA45 induced apoptosis and autophagy was medicated by the PI3K/Akt/JNK/p53 pathway.
To further demonstrate antitumor activity of CAA45, the tumor xenograft model bearing A549 cancer cell in BALB/c nude mouse was established and evaluated its anticancer activity in vivo. CAA45 at 5 or 10 mg/kg was able to reduce the tumor growth relative to the vehicle group. However, a slight body weight gain was observed among treat groups including the CPT treatment group as compared to the vehicle group, which may due to the fact that CAA45 may have some toxicity, similar as CPT. Further studies will be conducted to disclose the causes of the slight body weight gain.
In conclusion, the present study indicated that CAA45 potently in-hibited lung cancer proliferation with good selectivity over normal cells. CAA45 exerted its anticancer eﬀect by multiple mechanisms in-volving (i) inhibition of DNA Topo I activity, leading to DNA damage which results in cell cycle arrest at S phase; (ii) inhibition of cell mi-gration; (iii) induction of mitochondria mediated apoptosis and (iv) autophagy via PI3K/Akt/JNK/p53 pathway. Therefore, CAA45 could be considered as a promising lead compound targeting multiple im-portant pathways in cancer biology. Further studies to understand the molecular interactions and optimize the pharmaceutical properties of CAA45 are ongoing in our laboratory and the results shall be reported in due course.
Conflict of interest
The authors declare no conflict of interest.
This work was supported by the National Natural Science Foundation of China (No. 21572027, 21807008). We thank Yanfei Qiu and Hongmei Tian for technical assistance with animal studies.