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Antiproliferative effect of cryptotanshinone against human non-small cell lung cancer cells through inactivation of lncRNA HOTAIR /p-Akt signaling pathway
⁎Corresponding authors at: No.7, Yingxin Road, Jiancaoping District, Taiyuan 030003, China. fanyang2021@aol.com (Fan Yang), lima.oncology@yahoo.com (Li Ma)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Abstract
Cryptotanshinone is known as a quinoid diterpene extracted from the root of Salvia miotiorrhiza bunge and can show outstanding anticancer activities. In the present study, we explored the mechanism of action of cryptotanshinone, and its potential efficacy as a promising therapy for the treatment of non-small cell lung cancer (NSCLC) cell line (A549) in vitro. A549 cells were incubated with cryptotanshinone and cell proliferation and apoptosis were investigated using MTT, LDH, ROS, Annexin-V-FITC assays. The expression levels of lncRNA HOTAIR and p-Akt were quantified by quantitative real-time PCR (qPCR) and western blot, respectively. The results showed that cryptotanshinone mitigated cell proliferation in a dose and time-dependent manner through a significant increase in LDH release, ROS production, and apoptosis in A549 lung cancer cells. Treatment of A549 cells with cryptotanshinone also led to downregulation of lncRNA HOTAIR and protein level of p-Akt. Furthermore, the anticancer effect of cryptotanshinone was inhibited in the presence of NAC as a potential antioxidant. In general, it was found that cryptotanshinone is an effective agent in preventing lung cancer cells growth through downregulation of HOTAIR and p-Akt expression which is partially due to high level of ROS.
Keywords
Cryptotanshinone
Human non-small-cell lung cancer
lncRNA HOTAIR
1 Introduction
Lung cancer is the most common cancer of both sexes worldwide and more than 80% of patients with this cancer die within 5 years of being diagnosed (de Groot and Munden, 2012). The death rate from breast and prostate cancers is generally lower than the number of lung cancer victims (de Groot and Munden, 2012). More than 90% of lung cancers are caused by changes in the cells of the basal epithelium of the lungs and the lining of the lungs (Goldstraw et al., 2011). Lung epithelial cancers have four main cell groups as following: small cell lung cancer (SCLC) and non-small lung cancer (NSCLC) with different sup-types of adenocarcinoma, squamous cell carcinoma, and large cell carcinoma (Zappa and Mousa, 2016). SCLC accounts for about 15 to 20% and NSCLC about 80 to 85% of lung cancers (de Groot and Munden, 2012; Goldstraw et al., 2011; Zappa and Mousa, 2016). There has been little improvement in lung cancer treatment in the last 30 years (Lemjabbar-Alaoui et al., 2015). Smoking is a major risk factor for lung cancer because smokers are at risk for lung cancer about 20 times more than non-smokers (Hou et al., 2020).
Lung cancer has no symptoms in the early stages, and patients often were informed when the disease is in advanced stages (Cooley, 2000). Preventive measures such as avoiding pollutants and smoking and the absence of radon and asbestos gas in the environment are the best method of protection against the deadly disease of lung cancer (Xu et al., 1989).
Treatment patterns for lung cancer depend on the type of cancer, the condition at the start of treatment, age, general health, and how the patient responds to treatment (Smith et al., 1995). Lung cancer can usually spread easily to other parts of the body due to the rich blood supply and lymphatic system of the lungs (Laughney et al., 2020). Different therapeutic modalities such as surgery, chemotherapy, radiotherapy, photodynamic therapy, immunotherapy, and laser therapy have been widely used for treatment of lung cancer (El-Hussein et al., 2021; Zhang et al., 2018; Wirsdörfer et al., 2019). However, not only these methods can completely eliminate the malignant cancer cells, but also result in induction of some unwanted effects.
Despite many advances in cancer treatment, there is still a need to discover and introduce new and alternative or synergistic drugs due to the increasing emergence of mammalian tumor cell resistance to chemotherapy and its many side effects. There is a lot of evidence about the preventive properties of various types of plants which are consumed in the form of food, fruits, spices and vegetables against cancer (Xu et al., 1989; Smith et al., 1995; Laughney et al., 2020; El-Hussein et al., 2021; Zhang et al., 2018; Wirsdörfer et al., 2019; Abotaleb et al., 2020; Wei et al., 2019; Iqbal et al., 2017; Rayan et al., 2017; Chen et al., 2013; Maione et al., 2018; Yu et al., 2007; Lee et al., 2020; Shin et al., 2009; Jiang et al., 2017; Li et al., 2015; Zhang et al., 2018; Liu et al., 2020; Qi et al., 2019; Li et al., 2017; Yang et al., 2018; Li et al., 2020; Yu et al., 2017 Jun; Chen et al., 2015; Luo et al., 2020; Park et al., 2010; Chen et al., 2014; Wang et al., 2017; Le Li et al., 2020; Zhang et al., 2018; Jin et al., 2020; Chen et al., 2017; Ge et al., 2017; Ye et al., 2016; Chen et al., 2013) For this reason, secondary metabolites of plants as active herbal ingredients have been shown to reduce the risk of cancer (Abotaleb et al., 2020; Wei et al., 2019) more than 60% of anti-cancer drugs used today are derived from natural sources, ie plants, marine organisms and microorganisms (Iqbal et al., 2017; Rayan et al., 2017).
Cryptotanshinone is known as one of the major secondary metabolites can be extracted from the roots of Salvia miltiorrhiza Bunge widely utilized in traditional Chinese based-medicine for treatment of a number of disorders, including cancer (Chen et al., 2013), Alzheimer (Maione et al., 2018), stroke (Yu et al., 2007), Parkinson (Lee et al., 2020), and so on. Although, there are several reports on the anticancer effects of cryptotanshinone on different kind of cancers such as prostate cancer (Shin et al., 2009), ovarian cancer (Jiang et al., 2017), colorectal cancer (Li et al., 2015), breast cancer (Zhang et al., 2018), bladder cancer (Liu et al., 2020), and NSCLC (Qi et al., 2019) have been reported, some possible anticancer signaling pathways regulated by cryptotanshinone have not been fully addressed so far.
Recent reports indicate that non-coding RNAs (ncRNAs) play a key role in several cellular pathways (Li et al., 2017). Based on their transcript size, ncRNAs can be categorized into the small ncRNAs (sncRNAs, <200 bp) and long ncRNAs (lncRNAs, >200 bp) (Li et al., 2017). LncRNA molecule is encoded by HOX transcript antisense RNA (HOTAIR) gene on chromosome 12 (Li et al., 2017). It has been indicated that HOTAIR is unregulated in NSCLC cells (Li et al., 2017), which affects their tumorigenesis and metastasis (Li et al., 2017), drug resistance (Yang et al., 2018), and cell growth (Li et al., 2020; Yu et al., 2017 Jun). It has been reported that HOTAIR may control proliferation and invasion of cancer cells through Akt signaling pathway (Li et al., 2020; Yu et al., 2017 Jun). Despite the growing body of reports that HOTAIR and secondary metabolites compounds are involved in NSCLC proliferation, there are no reports on the relationship between cryptotanshinone and HOTAIR in NSCLC.
2 Materials and methods
2.1 Materials
Cryptotanshinone (purity > 98%, HPLC), dimethyl sulfoxide (DMSO), fetal bovine serum (FBS), RPMI-1640, and 3-(4, 5- dimethylthiazol2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma Aldrich (St. Louis, MO, USA). The stock solution of cryptotanshinone was freshly prepared in DMSO.
2.2 Cell culture
The human non-small cell lung cancer cells (A549) were cultured in RPMI 1640 medium containing 10% FBS, 1% penicillin and streptomycin at 37 °C in a humidified atmosphere of 5% CO2.
2.3 MTT and LDH assays
A549 cells were treated with increasing concentrations of cryptotanshinone (1–40 µM) and incubated for different times (12, 24, and 48 h). The supernatant was gently removed and the pellet was used for the MTT assay. Subsequently, a defined amount of MTT solution (5 mg/ml) was added to the cell medium and the samples were incubated for 4 h followed by addition of DMSO. The absorbance of the samples was then read using a microplate reader (Bio-Rad, Hercules, CA) at 570 nm. LDH assay was done using LDH assay kit (Sigma, USA) based on the manufacturer's recommended protocol and the absorbance was read at 450 nm.
2.4 Flow cytometry analysis for ROS and apoptosis assays
After treatment of the cells with IC50 concentration of cryptotanshinone for 48 h, the generation of intracellular ROS and the population of Annexin V-FITC positive cells were explored using ROS assay kit (ab113851, UK) and Annexin V-FITC apoptosis kit (ab14085), respectively using flow cytometry (BD Biosciences, San Diego, CA, USA).
2.5 Quantitative real-time PCR assay (qPCR)
A549 cells were incubated with IC50 concentration of cryptotanshinone for 48 h and the total RNA was isolated using Trizol (Gibco-BRL). cDNA was then synthesized using 15 ng of isolated RNA using relevant Synthesis Kit (Fermentas, Life Sciences, USA). Afterward, the expression level of HOTAIR was quantified using SYBR Green qPCR Master Mix (Fermentas, Life Sciences, USA) with an Applied Biosystems 7900HT Fast qPCR System based on specific primers for HOTAIR and GAPDH (Chen et al., 2015).
2.6 Western blot analysis
A549 cells were homogenized after incubation with IC50 concentration of cryptotanshinone for 48 h and the lysates were centrifuged at 15,000 rpm for 15 min. Afterward the protein concentration in the supernatant was quantified by BCA kit (Sigma, USA). Equal concentrations of isolated protein (30 μg/lane) were analyzed by SDS-PAGE, transferred onto polyvinylidene difluoride (PVDF) membranes, blocked in Tris-buffered saline, pH 7.6, 0.05% Tween 20, 5% non-fat dried milk, and incubated with specific monoclonal primary antibodies (1:900). After washing, the secondary antibodies (1:10000) conjugated with horseradish peroxidase (HRP) were added and incubated at room temperature for 50 min. Finally, the expression level of proteins was quantified using electrochemiluminescence (ECL) western blot detection reagents (Beyotime, China).
2.7 Statistical analysis
One-way ANOVA followed by Dunnett's multiple comparison test was used for analysis of different treatments using SPSS 20 software. Data were expressed as mean ± SD of three experiments, and P < 0.05 was considered statistically significant.
3 Results
3.1 Cryptotanshinone inhibit proliferation and increase the membrane damage of human A549 lung cancer cells over time
To explore the anti-proliferative effect of cryptotanshinone, human A549 lung cancer cells were treated with increasing concentrations of cryptotanshinone (0, 1, 5, 10, 20, 30, 40 μM) for 12, 24 and 48 h, and cell proliferation assay and membrane damage assays were determined using the MTT and LDH assays, respectively. The data indicated that cryptotanshinone significantly mitigated the proliferation of A549 cells (Fig. 1A) coincident with an increase in the LDH release (Fig. 1B) in a dose and time-dependent manner. Table 1 summarizes the IC50 concentrations in different incubation time determined by MTT assay.
| Time (hours) | IC50 (μM) |
|---|---|
| 12 | >40 |
| 24 | >40 |
| 48 | 18.53 ± 1.29 |
As tabulated in Table 1, it was observed that the IC50 concentrations of cryptotanshinone on A549 lung cancer cells at 12, 24, 48 h were > 40, >40, 18.53 ± 1.29 μM, respectively. Therefore, for conduction further experiments, the cells were incubated with IC50 concentration of cryptotanshinone (18.53 ± 1.29 μM) for 48 h.
3.2 ROS and apoptosis assays
High levels of ROS-induced by anticancer agents can result in activation of apoptosis. Therefore, we explored whether ROS is involved in the mechanism by which cryptotanshinone stimulates apoptosis in A549 lung cancer cells. N-acetylcysteine (NAC) is a potent antioxidant and if the presence of cryptotanshinone intensifies oxidative stress, the co-incubation of the cells with NAC can reduce the generation of ROS and subsequent apoptosis. As shown in Fig. 2A, B, the fluorescence intensity of DCF significantly increases in the cryptotanshinone-treated cells in comparison with the control group (***P < 0.001), whereas the co-incubation of cells with NAC reduced the fluorescence intensity of cells incubated with IC50 concentration of cryptotanshinone (***P < 0.001).
Furthermore, to investigate whether the anticancer effect of cryptotanshinone on A549 lung cancer cells was linked with the process of apoptosis, we assessed the apoptosis of the cells with annexin V-FITC apoptosis assay using flow cytometry. As shown in Fig. 2C, D, after exposure to cryptotanshinone for 48 h, the intensity of Annexin V-FITC (Fig. 2C) and the percentage of Annexin-FITC positive cells (Fig. 2D) significantly increased relative to the cells co-incubated with NAC (***P < 0.001).
3.3 Cryptotanshinone downregulate the expression level of lncRNA HOTAIR in human A549 lung cancer cells
We aimed to explore whether the expression level of HOTAIR were influenced upon incubation of A549 lung cancer cells with cryptotanshinone. The data demonstrated that cryptotanshinone significantly downregulated the expression level of LncRNA HOTAIR (Fig. 3), which was reversed in the presence of NAC.
3.4 Cryptotanshinone inactivate Akt in human A549 lung cancer cells
It has been well-documented that downregulation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway mitigate cell proliferation by inducing apoptosis. Therefore, we explored the phosphorylation level of Akt in cryptotanshinone -incubated A549 cells by western blot (Fig. 4A). As shown in Fig. 4A, B, it was observed that after treatment of A549 cells with IC50 concentration of cryptotanshinone, a significant reduction in the expression level of phosphorylated Akt occurs. This outcome revealed that downregulation of the Akt signaling pathway could play a key role in inhibiting cell proliferation mediated by apoptosis induction stimulated by cryptotanshinone (Fig. 4A, B).
4 Discussion
It has been shown that cryptotanshinone in traditional medicine can be used for preventive and anticancer purposes including a potential agent against hepatocellular carcinoma (Luo et al., 2020), prostate cancer (Park et al., 2010) and lung cancer (Chen et al., 2014). It has been also shown that cryptotanshinone as an active secondary metabolite can increase the potency of anticancer drugs against cancer cells previously were resistant to several chemical drugs (Wang et al., 2017) and reduce the unwanted effects of anticancer drugs against normal cells (Le Li et al., 2020). Although the anticancer effects of cryptotanshinone against different cancer cells have been explored in detail, its effects on the apoptosis of human NSCLC and the associated mechanism through ncRNAs have not been well-reported. Current studies have reported that cryptotanshinone was active against lung cancer cells and the anticancer effect of cryptotanshinone against these cells were attributed to the induction of apoptosis through different mechanisms, including IGF-1R/PI3K/Akt (Zhang et al., 2018); microRNA-146a-5p/EGFR axis (Qi et al., 2019); and promoting TAZ translocation from nuclear to cytoplasm (Jin et al., 2020).
The present study showed that downregulation of p-Akt at protein level and lncRNA HOTAIR could improve the anticancer effects of cryptotanshinone against lung cancer cells mediated by high levels of ROS. Indeed, it has been unknown the impacts of lncRNA HOTAIR and the association between lncRNA HOTAIR and p-Akt in lung cancer. In this study, it was shown that the effect of lncRNA HOTAIR and the correlation between lncRNA HOTAIR and p-Akt in human NSCLC is regulated by cryptotanshinone through generation of high levels of ROS.
These outcomes are consistent with the reports of previous studies indicating the participation of ROS in killing of lung cancer cells (Chen et al., 2017; Ge et al., 2017).
Previous studies have determined that cryptotanshinone triggers cytotoxicity by the stimulation of apoptosis through a caspase-dependent pathway via mitochondria (Ye et al., 2016; Chen et al., 2013).
We showed that cryptotanshinone could stimulate the apoptosis of lung cancer A549 cells. The apoptotic effect of cryptotanshinone was confirmed by the flow cytometry analysis. In addition, NAC significantly rescue the apoptosis triggered by cryptotanshinone, indicating that this effect of cryptotanshinone on induction of apoptosis of A549 cells is partially dependent on the downregulation of lncRNA HOTAIR and p-Akt signaling pathways via the generation of ROS, while other pathways are also involved in the anticancer effects of cryptotanshinone.
Clearly more detailed in vivo investigations on the molecular mechanism of action of cryptotanshinone is vital for the fully exploring the effect of cryptotanshinone on apoptosis of lung cancer A549 cells.
5 Conclusion
The present study showed that cryptotanshinone could increase the apoptosis of lung cancer A549 cells, and indicated the role of ROS in downregulation of p-Akt/lncRNA HOTAIR. A more detailed study about the mechanism of action of cryptotanshinone could efficiently improve clinical development of this compound for the treatment of lung cancer.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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