Autophagy-Inducing Effect of Compound Berberine on CNE2 NPC Cells via Interference with the Targets in P13K/AKT/mTOR Signaling Pathway

Objective: To investigate the autophagy-inducing effect of Compound Berberine (CBBR) on CNE2 nasopharyngeal carcinoma (NPC) cells and its possible targets in P13K/AKT/mTOR signaling pathway. Methods: CNE2 cells at exponential growth phase were taken as the target cells in this study. Firstly, IC50 concentration for CBBR was determined by MTT assay. Then, 3 different concentrations of CBBR, 0.25 mg∙mL −1 , 0.50 mg∙mL −1 and 1.00 mg∙mL −1 , around the concentration of IC50, were taken for followed intervention experiments respectively. Fluorescein labeling method was utilized to assay the inducing effect of CBBR on the autophagic activity of CNE2 cells, followed by Western blot procedure to explore the changes of key messenger molecules in the autophagy-related signaling pathway of P13K/AKT/mTOR, both combined with 3-MA block test in a comparative way and carried out by detecting the expressive levels of Beclin 1, LC3-II and LC3-I as well as the ratio of LC3-II:LC3-I. Results: IC50 of CBBR was determined at the level of 0.5 mg∙mL −1 . The inducing effect of CBBR on autophagy of CNE2 cells was shown occurring in various modes, not a simple concentration-dependent tendency, with its effect minimal at the concentration of 0.25 mg∙mL −1 and maximal at the concentration of 0.50 mg∙mL −1 , while only slightly higher at the concentration of 1.00 mg∙mL −1 than that of 0.5 mg∙mL −1 . Although its inducing effect was weakened a little following the pretreatment by 3-MA, the effect combined with CBBR was still significantly higher than that of simply blocked by 3-MA. Moreover, changes in the expressive levels of Beclin1, LC3-II and LC3-I as well as LC3-II:LC3-I all showed a tendency corresponding to the changed autophagic features of CNE2 cells (P < 0.05 or P < 0.01), given more supporting evidences for the effect of CBBR on autophagy of CNE2 cells. Conclusions: CBBR can bring about inhibiting effect on the proliferating activity of CNE2 cells through inducing increased autophagic activity via intervening targets in P13K/AKT/mTOR signaling pathway, and this effect could not be completely blocked by the antagonist 3-MA.

Autophagy is a kind of inherent endogenous mechanism for self-degradation of sub-cellular fractions within all kinds of eukaryotic cells [17], being a kind of phenomena with highly conservative characteristics within living organisms including xenophagy [18] and being crucial for the quality control mechanisms and maintenance of cellular homeostasis in various stem cells as well [19]. Under some stress conditions such as hunger, eukaryotic cells can survive by necessary energy supply guaranteed through such a very important biological event of "self-digestion" to maintain a continuous process of intracellular material metabolism in a turnover way, during which proteins and other components of damaged, senescent and dead organelles are degraded by enzymatic degradation of lysosomes and then being transformed into energy supply to the cells to help themselves get through the crisis caused by such stress stimulating events. This kind of mechanism can also be utilized to block damage and apoptosis inducing effects on cells themselves by degrading some toxic substances within them. Therefore, this mechanism has been well known as cellular "sweeper" or "cleaner". Autophagy can widely occur in various kinds of pathophysiological processes among eukaryotic cells, including developing, aging, neurodegenerative disorders, carcinogenesis and some kinds of infectious diseases. So, autophagy is closely associated with the several development processes and many kinds of diseased progress. However, autophagy holds a special feature of "dual-ity" at some stages of illness or under some stress conditions, just like a rapier or "double-edged sword". In this aspect, carcinogenesis can be taken as the most typical sample associated with such an effect, that was discovered earliest. Increasing evidences are showing that autophagy plays very important roles in the process of tumorigenesis and in the response of tumor cells to anti-tumor therapies so that research in this field is becoming one of the hot subjects.
It has been confirmed that abnormality in autophagy can lead to transformation of normal cells into malignant ones via inducing gene mutation. Meanwhile, abnormal changes in the mechanisms of autophagy may induce, even exacerbate, the resistance of tumor cells to anti-tumor therapies and lead them to escape from the attack of anti-tumor therapies. On the other hand, synthesized therapeutic efficiency of anti-tumor therapies may be improved largely once tumor cells are successfully transformed into the process of apoptosis, or necrosis, via the pathway of autophagy inducing effect to blocking its protective effect on the survival of tumor cells.
Based on such a consideration, this work was aimed at exploring the intervening effect of Compound berberine (CBBR) on autophagic activity of NPC cells and its associated mechanism possibly with the signaling pathway of autophagy, because it has been confirmed through a series of previous work that CBBR held very strong inhibitory effects on the proliferating activity of NPC

Methods
1) All cell cultures were carried out routinely in an incubator with 5% CO 2 at 37˚C and saturated humidity.
2) Inhibitory effect of CBBR on the proliferating activity of CNE2 cells CNE2 cells at exponential growth phase in the culturing system of 1640 medium, supplemented with 10% fetal calf serum, were prepared into single cell suspension and inoculated in 96-well culture plates at a density of 5 × 10 3 cells/mL. Following 24 h routine culturing, culture medium was exchanged with CBBR 1640 medium to culture for another 24 h, with the final CBBR concentration being 0.25, 0.50, 1.00, 2.00 and 4.00 mg•mL −1 respectively and equivalent to such quantities of crud drugs, with 5 paralleled wells for each concentration and a blank control in all these tests. Then, medium was displaced with 150 μL 0.5% MTT solution for a culturing period of 4 h. Followed were the steps to discard MTT medium and to add DMSO solution at a volume of 150 μL/well, with culture plates transferred to a shaking table for 10 min under a shaking status at 50 r/min. After this step, OD value of each well was detected on a Fully Automatic Enzyme Label Analyzer at the wave length of 490 nm for calculating cellular inhibitory rate and IC50 concentration of CBBR. All the experiments were repeated in triplicate.
Inhibitory rate was calculated based on the following equation where OD was the abbreviation of optical density, ODc was the OD value of control group, ODe was the OD value of experimental group.
3) Autophagy-inducing effect of CBBR on CNE2 cells supplemented with 3-MA blocking test. a) Autophagy-inducing effect determination CNE2 cells were inoculated in 6-well culture plates with a density of 1 × 10 5 cells/well, cultured for 24 h in 5% CO 2 incubator at 37˚C with saturated humidity. Then, cells at exponential growth phase in this culturing system were designated into three interfering groups with 3 different CBBR concentrations, i.e. 0.25, 0.50, 1.00 mg•mL −1 selected on the basis of its IC50 concentration, i.e. the concentrations at IC50, less than doubled the IC50 and twice the IC50 respectively. By the time of medium exchanging, all wells in interfering groups were added with CBBR medium at their corresponding concentrations in a total value of 2 mL in each well for a continuing culture period of 24 h, with a paralleled blank control group set as well. By the end of interfering culture period, medium in all culture wells was discarded. Following 2 times washing with PBS, added was the solution of 50 μM MDC into each well for a further culture of 1 h. Then, plates were observed un-der an inverted fluorescence microscope to determine the autophagy inducing levels among different CBBR concentrations on CNE2 cells after the medium in each well being discarded and washed for 3 times with PBS. The experiment was also repeated in triplicate. b) Blocking test on autophagy-inducing effect of CBBR Based on the determination of autophagy-inducing response as tested above, cells at exponential growth phase were inoculated into 6-well plates at a density of 1 × 10 5   i) Preparation of cell lysis solution. CNE2 cells were inoculated in 100 mm culture dishes at a density of 7 × 10 5 cells/mL, cultured in a 5% CO 2 incubator at 37˚C with saturated humidity till cells growing to an exponential growth phase.
By this time, cells were designated into 3 interfering groups to be treated with the concentrations of CBBR at 0.25, 0.50, and 1.00 mg•mL −1 , and added with CBBR medium at corresponding concentrations respectively and with a paralleled blank control group designated as well. Then, cells were continuously cultured for 24 h. After that time, medium in each well was discarded, cells were collected from each group routinely in an ice box for 15 min, supplemented with International Journal of Clinical Medicine 50 μM RIPA lysis solution containing 1% PMSF to produce cell lysis, and followed by cells washed 3 times with cold PBS respectively. After this step, cell lysis solutions were transferred into 1.5 mL PE tubes for centrifugation at 4˚C and 12,000 rpm for 10 min, with the supernatants collected respectively by the end of this operation for following procedures, also repeated in triplicate.
ii) Determination of protein concentration. This step of operation was carried out following the instruction of operating manual of BCA protein quantitative detection kit, with BCA working solution and standard diluent prepared to draw a standard curve. Following the sample of each group loaded, they were detected on a microplate reader at 570 nm to calculate the protein concentration of each sample respectively by referring to the standard curve.
iii) Preparation of electrophoresis gels. SDS-PAGE electrophoresis gels were prepared according to the routine standard operation program. iv) Electrophoresis. A total of 25 μL sampling volume was loaded into one well of chamber for each sample. Then, electrophoresis was carried out at a voltage of 80 V with a maximal current. When target proteins swimming to the distance larger than 1cm to the lower edge, the electrophoresis was ended.
v) Protein transmembrane. Once gels were taken out of the electrophoresis groove, they were appressed closely to the PVDF membrane and transferred into a diaphragm groove for 60 min at a 300 mA constant current. (CMG) respectively. By the time of medium exchanging, cells in the dishes of MG and CMG were pretreated with 2 mM 3-MA for 1h at first, and then, exchanged were CBBR medium at IC50 concentration into the dishes of CG and CMG and normal medium into the dishes of BG and MG for a continued culturing of 24 h respectively. After this step, followed were cell collection, cell lysis solution preparation, determination of protein concentration, electrophoresis, protein transmembrane, antibody incubation, development and image analysis, and the determination on the expressive levels of the key molecules in the signaling pathway of PI3K/AKT/mTOR in these cells respectively, as the same in (1) of 1.2.4. Also, this series of experiment was repeated in triplicate.

Statistical Processing
SPSS21.0 statistical software (IBM, Chicago, IL, USA) was utilized to the analysis of these experimental data. One-way ANOVA and Dunnett's multiple comparisons test or Tukey's multiple comparisons test were performed in this process for various cases respectively. Results were reported as mean ± SD. Data were considered significant statistically when P ≤ 0.05.   Figure 1).

Autophagy-Inducing Effect of CBBR on CNE2 Cells
It was shown that the number of autophagosomes increased gradually with cor- being not a proportional relation between the applied concentrations and the effect potencies (see Figure 2 and Figure 3). Once pretreated by autophagy-blocking reagent 3-MA in the culturing system, displayed was still relatively stronger inducing effect on autophagic activity as compared with that of blank  controlling group and negative controlling group just without 3-MA treated, while the active strength of autophagy-inducing effect was shown some degrees of decrease when compared with that without 3-MA treatment. These data meant that the autophagy-inducing effect of CBBR could not be completely blocked by 3-MA. Moreover, it could be found further that cells in the group pretreated by 3-MA and following 0.5 mg/mL CBBR treatment still showed a significantly stronger autophagy activity when compared with that in the group of cells simply treated by 3-MA alone, though its autophagy-inducing effect declined at some level, even being not at an obviously blocked status as shown in Figure 4 and Figure 5. All these suggested that the autophagy-inducing effect of CBBR on CNE2 cells could not be completely blocked by 3-MA, showing a kind of different effective mechanism at some levels with that of classic pathway, perhaps another unknown pathway(s) for such a phenomenon present here.

The Effect of CBBR on the Expressive Activity of Autophagy-Specific Proteins of CNE2 Cells
Beclin-1 and LC3 are the specific protein markers of autophagy, showing a positive proportional relation with their expressive activities as well as the ratio of LC3II to LC3I with the autophagic level. Especially, there was a similar proportional increase in the protein fragment of LC3II as shown in the electrophoresis strip and the level of autophagy. Therefore, the level of LC3II:LC3I ratio can be taken as the key index to estimate the strength of autophagic activity. As shown in Figure 6, the expression of Beclin-1 and LC3II were significantly elevated (P < 0.05 -0.01) in CNE2 cells following the treatment of CBBR for 24 h at various concentrations as shown in Figure 6, with the maximal expressive levels being Beclin-1 and LC3II proteins as well as the highest peak value of LC3II:LC3I ratio about 5.1 in the group of cells treated by the concentration of 0.50 mg•mL −1 CBBR (P < 0.01). Furthermore, the rank of change in the size of ratio was in   Among these groups of blocking experiment, the strength of autophagy-inducing effect, as indicated by the ratio of LC3II:LC3I, was ranked in this order of CBBR intervening group not treated by 3-MA, CBBR intervening group pretreated by 3MA, the group simply treated by 3-MA, and the blank controlling group from the higher to the lower, with no significantly statistical significance present between the last two groups (P > 0.05), while the most prominent strength of autophagy-inducing effect was determined in the group of cells intervened with CBBR alone but not treated by 3-MA, significantly higher than that of all the other groups. Although the inducing effect of the group pretreated by 3-MA followed by CBBR treating (CMG) was obviously lower than that of the former, it was still higher than that of simply treated by 3-MA, see (Figure 7).
These data suggested that 3-MA could not completely block the autophagy-inducing effect of CBBR on CNE2 cells again, and CBBR might bring about autophagy response in CNE2 cells via other pathways in part.

The Effect of CBBR on the Activities and Phosphorylation Levels of the Key Messenger Molecules in PI3K/AKT/mTOR Signaling Pathway of CNE2 Cells
Treatment with different concentrations of CBBR could bring about somewhat different characteristic effect on the activities of the key messenger molecules in PI3K/AKT/mTOR signaling pathway of CNE2 cells. Taking the internal reference as the baseline to analyze the corresponding changes in the activities and their phosphorylation levels of the key molecules in PI3K/AKT/mTOR signaling pathway of the cells treated by CBBR for 24 h, it was shown, when compared with that of controls, all groups of cells intervened by CBBR showed a down-regulated tendency in the activity levels of PI3K, AKT, p-AKT and p-mTOR (P < 0.05 or P < 0.01), indicating that significantly declined were the expressive and/or phosphorylation levels of the key molecules in this signaling pathway, with an obviously responsible feature showing a concentration-dependent manner for PI3K and p-AKT to CBBR intervening, i.e. the effect variation ratio being maximal at the highest concentration of 1.00 mg•mL −1 CBBR, followed by a decreased changing tendency in the effect variation ratio following the decline of CBBR concentration. However, the features of change in the response of AKT and p-mTOR showed a super-optimal concentration manner, i.e. their activity levels reached at the maximum at the concentration of 0.50 mg•mL −1 , almost similar with that of specific autophagic index LC3II:LC3I ratio response determined by Western blot assay. These data might suggest that there should be possible for different messenger molecules in the same signaling pathway varying in their response manner to a same intervening factor. Therefore, it should be possible that there might be other no classic signaling pathways present for autophagy inducing besides that could be blocked by 3-MA, as seen in Figure 8 and Table 1.

Discussion
It has been confirmed widely that berberine holds very strong antitumor effect for long time via such effects as inhibiting cell proliferation activity, blocking cell cycle at G2 phase, inhibitory effect on the synthesis of DNA and protein, suppressing the biological synthesis response through integrating TOPI at S phase of the cell cycle, inducing apoptosis, repressing the potency of migration and International Journal of Clinical Medicine  and to activate T cell mediated cellular immune function [24]. The present work is mainly focused on the exploration of autophagy-inducing effect brought about by CBBR on CNE2 cells and its main mechanism of this kind of effect.
There was a different responsiveness present for CBBR between its cell proli- Blocking test could provide still more reliable information to explain many complex results in the research of biological mechanisms as found in this work.
Therefore, autophagy-blocking tests were set up here. As also seen here, there was no autophagic response found in the blank controlling group, and occasio- Meanwhile, the ratio level of LC3-II:LC3-I can be taken as the specific index to evaluate the trend in the intensity of autophagy since the change of LC3-II in its expressive level was paralleled to the forming rate of sub-cellular autophagic structures. As shown in the interfering test with CBBR and blocking test with 3-MA, the expressive levels of LC-3 were both significantly elevated, with corresponding LC3-II/LC3-I ratio increased obviously as well. When taken such a ratio as the indicator to evaluate the autophagic activity in these experiments, the intensities of autophagic response could be ranked in such an order from the higher to the lower as the group of cells interfered with CBBR alone, the group of cells interfered with CBBR plus 3-MA pretreatment, and the blank group. This trend in the intensity change of autophagic response was extremely similar with that reflected by the morphological indicators of autophagy from the results of autophagy-blocking test, all showed a phenomenon suggesting that autophagy blocker 3-MA could not completely block the autophagy-inducing effect of CBBR on NPC cells. Once again, it should be possible that there would be other nonclassical pathway(s) present for CBBR to induce intensive autophagic response in NPC cells other than the classic one. It should be worth exploring in future.
The mechanism of autophagy for CNE2 cells might involve in the signaling pathway of PI3K-Akt-mTOR and their phosphorylation levels. As shown in Table 1, these cells displayed obviously elevated expressive levels of PI3K and p-AKT in a concentration-dependent mode, increasing with the elevation of CBBR concentration to the highest peaks of value at 1.00 mg•mL −1 , while the peak expressive levels of AKT and p-mTOR were seen at such a CBBR concentration of 0.50 mg•mL −1 . These effective features indicated that they were kept in a change trend similar with that of specific autophagic proteins, meaning that this pathway should be involved in the initiation of autophagy and its speeding up in signaling transduction.
However, there were some other special features shown from these experiments for the effect mode of CBBR on mTOR. As shown from the relative ratio of background activity of mTOR, the value at this level was 0.96, while it reduced to 0.88 at lower CBBR concentration of 0.25 mg•mL −1 showing an inhibitory effect significantly lower than its background activity. However, it elevated obviously when the concentration of CBBR increased to 0.50 mg•mL −1 and then, got to a peak value of 1.30 at the concentration of 1.00 mg•mL −1 , also appeared a similar trend in the change of activity with that of PI3K [26]. Evidently, this phenomenon was closely associated with the effect of mTOR as a negative regulatory factor on autophagic signaling pathway, involved in the initiation of mTOR biosynthesis to inhibit autophagy, or it could initiate autophagy [27]. Such a special reactive mode of mTOR to the interfering of CBBR further meant that there would be other possible underlying mechanism present and was also consistent with the finding of the autophagy caused by 11-MT that was found to be brought about via activation of the AMP activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) and the c-Jun N-terminal kinase (JNK) signaling pathways [28].
Based on the discussion above, we could deduce that one of the important mechanisms for CBBR to inhibit the proliferating activity of CNE2 cells might be achieved through activating their autophagic response, as that of quercetin to promote hepatoma cells going into apoptotic program via inducing autophagy at first [29]. Meanwhile, curcumin could induce apoptosis in malignant mesothelioma cells through initiating autophagy flow so as to suppress their level of proliferation [30]. Some ingredients of herbal medicine, such as TanshinoneI (TSI) and Aspidosperma alkaloids, held anticancer potential against human glioma U87 MG cells and could reverse the resistance of chemical drugs against cells of ovarian cancer via inducing apoptosis and autophagy through ER stress and AKT signaling induced by TS I via intracellular reactive oxygen species accumulation [28] [31], though some reports indicated that autophagy was significantly associated with tumor grade and EMT, conferring the survival advantage to neoplastic cells to anti-cancer therapies and significantly affecting the invasive potential of cancer cells and supports their metastatic dissemination in a tissue and tumor stage dependent manner [32] [33]. However, it has been confirmed recently that PHLPP2 was able to mediate BECN1/Beclin1 stabilization indirectly to promote BECN1-dependent macroautophagy/autophagy to inhibit BC tumor cell growth and that increased autophagy via attenuating MIR516A resulted in a dramatic inhibition of xenograft tumor formation in vivo [34].

Conclusion
In summary, one of the main effective characteristics of CBBR on NPC cells should be prominently the autophagy-inducing effect, one of the important mechanisms for CBBR to inhibit, even to kill, tumor cells through which the proliferating activity of CNE2 cells can be inhibited significantly, even though autophagic response could induce various, even contrary, effects on different kinds of tumor cells [35] [36].