A study in the effect of Oridonin on the lung cancer therapeutic effect of lentinan


 Oridonin has been shown to have a potency for the improvement of the antitumor effect of lentinan (LNT). In this study, we tested the effect of oridonin, LNT, and the combination of them on a lung cancer. The study demonstrated that oridonin enhanced the antitumor effects of LNT and is conducive to the development of oridonin and LNT as a novel cancer drug regimen.


Introduction
In recent years, traditional medicine has been studied for clinical treatment for their low side effects (1,2).
To achieve fewer side effects, increasing attention has been given to a fungal polysaccharide, lentinan (LNT), for its strong antitumor activity (3,4). It was reported to effectively affect division, differentiation, growth, and senescence of cells (5). LNT was reported to effectively prevent patients from cancers caused by chemical or viral carcinogens. In clinical, LNT enhances chemotherapy and improves the survival of patients in several cancer types, including gastric, colon, breast, and lung cancers. Current evidence also showed that it targeted small-cell lung cancer cells (6). Although it has a relatively weak effect on cancers, it is a promising chemotherapy synergist.
H group (0.2 ml/day of 20 µg/ml oridonin and 0.2 ml/day of 300 µg/ml LNT). The dose was converted from the clinical dose for human patients. For the rst set of animal experiments, after 10 days, the busts of the mice were measured with a mini tape measure before and after the treatment, and the bust increased over 50% compared with the size before injection was de ned as signi cant lung cancer metastasis. All the mice were euthanized followed by the collection of lung tissues. The method of euthanasia used at end point was CO 2 inhalation. The euthanasia chamber enabled animals to be readily visible which provided a minimum purity for CO 2 of 99.0%. This allowed unconsciousness with minimal distress to the animals. The lung tissues were used to extract mRNA and protein for the PCR and the Western Blotting assay. For the other set of experiments, the mice were fed until the endpoint to obtain the survival rate. The criteria for the endpoint were: 1) tumor growth that impedes the ability to ingest food or water; 2) the tumors pain or distress that cannot be relieved with palliative measures; 3) Solid tumors estimated to exceed 20% of normal body weight. The death of animals was recorded every 5 days.

Statistical analysis
All experiments were repeated at less three times. One-way ANOVA with post-hoc Tukey's tests was used to analyze the difference, P < 0.05 was considered to indicate a statistically signi cant difference. SAS v9.1 statistical software package (SAS Institute Inc., Cary, NC, USA) was used for the statistics.

Results
The effect of oridonin and LNT on the growth of MRC-5 and A549 cells Cell viabilities were determined using MTT assay. The viabilities of MRC-5 and A549 cells were not affected by 0-20 µg/ml oridonin. 0-300 µg/ml LNT did not affect the viability of MRC-5 cells, but 50-400 µg/ml LNT signi cantly inhibited the viability of A549 cells. The oridonin, LNT, or the combination of both did not affect MRC-5 cell viability. The oridonin had no effect on A549 cell viability but LNT signi cantly suppressed A549 cell viability, and the used of oridonin increased the suppression effect of LNT on A549 cells. 20 µg/ml oridonin was used in the subsequent study because it was the highest concentration that had no signi cant effect on cell viability. In addition, 300 µg/ml LNT was selected for the subsequent study as a high concentration group (LNT-H) because it had the strongest potency in the viability of cancer cells while had no effect on the viability of normal cells. As a comparison, we also used a low concentration group (LNT-L) which was 100 µg/ml LNT. The effect of oridonin and LNT on mRNA and protein expression of the p53/p21 pathway proteins in A549 We suggested the viability effect was associated with p53/p21 signaling, thus we also tested them. LNT at both concentrations tested increased the mRNA and protein expression of p53 and p21 in A549 cells. Oridonin further increased the mRNA and protein expression of p53 and p21 in LNT treated A549 cells.
These results suggested that the p53/p21 pathway was involved.
The effect of oridonin and LNT on mRNA and protein expression of NF-κB and IκB-α in A549 Additionally, LNT at both concentrations tested increased the mRNA and protein expression of NF-κB in A549 cells with a more dramatic increase in LNT-H. Oridonin further increased the mRNA and protein expression of NF-κB in LNT treated A549 cells. On the other hand, LNT at both concentrations tested decreased the mRNA and protein expression of IκB-α in A549 cells with more decrease in LNT-H. Oridonin further decreased the mRNA and protein expression of IκB-α in LNT treated A549 cells. These results suggested that the NF-κB and IκB-α signaling was involved.
The effects of oridonin and LNT on lung tumor metastasis in mice We established lung cancer metastasis mice model and treatment them with LNT-L, oridonin, oridonin + LNT-L, LNT-H, and oridonin + LNT-H. Mice without cancer or any treatment and mice with lung cancer but any treatment were used for comparison. After 10 days, the oridonin alone had no effect on short time lung cancer metastasis. LNT treatment decreased the metastasis with a higher inhibitory rate in LNT-H group than in LNT-L group. Oridonin improved the suppression of LNT against lung cancer metastasis in both groups. These results suggested that oridonin help LNT decrease lung tumor metastasis in mice.

The effects of oridonin and LNT on the overall survival of mouse with lung tumor cells
We also conducted a survival assay to test the effect of oridonin and LNT. Results showed that oridonin alone had almost no effect on the survival of the animals. Both LNT-L and LNT-H improved the survival and LNT-H had a better outcome than LNT-L. Oridonin increased the improvement effect of LNT on the survival rate. Results indicated that the combined use of both oridonin and LNT optimized survival.
The effects of oridonin and LNT on the mRNA and protein expression of caspase-3, caspase-8, and caspase-9 in mice lung tissues After the lung cancer cell injection, the mRNA and protein expression of caspase-3, caspase-8, and caspase-9 signi cantly decreased in lung tissue samples. The LNT at both concentrations tested increased the mRNA and protein expression of caspase-3, caspase-8, and caspase-9 in lung tissue samples with a more signi cant increase in LNT-H. Oridonin further increased the mRNA and protein expression of caspase-3, caspase-8, and caspase-9 in the lung of LNT treated animals. These results were similar to those in A549, which con rmed the effect of oridonin and LNT on lung cancer.

Discussion
The signi cance of this work is to help explain the treatment effect of oridonin and LNT in clinical lung cancer patients in our hospital.
Our results showed that lung cancer cell line A549 was much more sensitive than normal human fetal lung broblast cell line MRC-5 toward LNT. This suggested that LNT can be a potential cancer medicine with low side effects. LNT was proved to have a suppression effect on some cancer cells (19) as well as an immunomodulatory effect on cancer patients (20). A retrospective study demonstrated that LNT improved the quality of cancer patients' life and remarkably promoted the e cacy of chemotherapy and radiation therapy during the cancer treatment (21) An in vivo study also revealed that LNT showed therapeutic potential for colitis-associated cancer. While for the oridonin, previous studies showed that 36 μg/ml (0.1 mmol/l) oridonin inhibited breast cancer growth and metastasis through blocking the Notch signaling (7).
In this study, we used the MTT assay to test the viability of lung cancer cells. The MTT assay was widely used in cancer pharmacological studies (22). Surprisingly, our results show that 0-300 μg/ml oridonin had little effect on both MRC-5 and A549, indicating that lung cancer might have lower sensitivity than breast cancer toward oridonin. In addition, oridonin was also shown to inhibit human pancreatic cancer migration (8). The survival rate is a critical indicator of cancer therapy in different cancer types (23).
Thus, we mimic the clinical treatment and survival in the mice as a previous study (24). The in vivo evidence of this study showed that oridonin along failed to suppressed the migration of lung cancer cells and did not affect the survival of mice with lung cancer. However, we found that oridonin promoted the effects of LNT both in the viability of A549 and the migration and the survival of mice. These results indicated that oridonin might trigger pathways that facilitate the actions of LNT. However, in survival experiment, we applied inhalation anesthesia. The anesthesia was reported to potentially affects cancers (23,25), this might potentially affect the results.
To explore the underlying mechanisms, we tested several potential targets of LNT in both A549 and lung tissue samples. Studies show that proliferation and apoptosis affected cancer viability (26). We suggested that apoptosis might be involved in the effects of oridonin and LNT. Firstly, we looked at the caspase signaling pathway. Caspases, cysteine-aspartic proteases, cysteine-dependent aspartate-directed proteases, are a family of protease enzymes playing essential roles in programmed cell death and in ammation (27). A previous study showed that the co-treatment with paclitaxel and lentinan enhanced cell apoptosis rate by inducing caspase-3 activation (28). Here we discovered that LNT suppressed A549 cell viability by improving the expression of apoptosis executioner caspase-3 and the oridonin could further promote this improvement. Moreover, we also found potential negative feedback of the Caspases signal, since the expression of apoptosis initiator in Caspases, the Caspases-8 and 9, increased with Caspases-3.
In fact, lentinan was reported to exert synergistic apoptotic effects with paclitaxel in A549 cells (28). The apoptosis-inducing effect of lentinan was also reported in a study in human bladder cancer T24 cells (29). In hepatoma cells, oridonin was proved to promote the effects of lentinan through regulating apoptotic genes. To con rm that these effects are mediated by apoptosis (28), we further tested an apoptosis regulatory pathway, the Bax pathway. BCL2 family members act as anti-or pro-apoptotic regulators for cancer cells. Bcl-xL acts as an anti-apoptotic protein by preventing the release of mitochondrial contents such as cytochrome c, which leads to caspase activation and ultimately, programmed cell death (30). In the present study, the expressions of Bax, Bcl-2, and Bcl-xL were affected by LNT and oridonin, indicating that the effect of them was mediated by apoptosis.
The expression of the BCL2 family genes is regulated by the tumor suppressor p53 and has been shown to be involved in p53-mediated apoptosis (31,32). We proposed that p53 and p21 might be the upstream targets of LNT and oridonin. Hence, the expression of p53 and p21 in A549 and lung tissues were determined, and it came out that the expressions of p53 and p21 were improved by LNT and oridonin. NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells, is a protein complex that controls transcription of DNA, cytokine production and cell survival while IκBα, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha, is cellular proteins that function to inhibit the NF-κB transcription factors (33). The present study proved that NF-κB and IκBα are involved in the effects of LNT and oridonin on cancer, and their expression changed and played a role in the decrease of the cell viability. In the lung tissues, the result was similar to that of cancer cells, which con rmed the conclusion under in vivo condition.
Our study demonstrated that oridonin enhanced the antitumor effects of LNT. We also found several potential regulatory targets of the effect of oridonin and LNT. However, there are other mechanisms that might be involved, such as cancer stem cells (34), ion channels in cancers (35)(36)(37), etc. Besides, studies have showed that traditional medicines(38) and anesthetics (23,24,37) might affect cancer treatments. Hence, more work is required in the future. Recently, many compounds derived from traditional medicines were explored for potential clinical use (39)(40)(41). In Cancer treatment, although traditional medicines were not able to cure cancer alone, they are applied in the combination of medical therapy for reducing the adverse effects caused by chemotherapy or radiotherapy, thus improving therapeutic outcome and quality of life for patients (42). Here we tried to develop a compound from a traditional herb medicine to be a potential chemotherapy synergist. This study is conducive to the development of oridonin and LNT as a novel cancer drug regimen and contributes to the application of traditional medicine in clinical treatments.

Declarations Con ict of Interest
There is no con ict of interest.