Physalis alkekengi var. franchetii extracts exert anti-tumour effects on non-small cell lung cancer and multiple myeloma by inhibiting STAT3 signalli


 Background: Physalis alkekengi var. franchetii is an herb that possesses various ethnopharmacological applications. Herein, our current study focuses on the anti-tumour effect of the combination of various physalins, which are regarded as the most representative secondary metabolites from calyces of Physalis alkekengi var. franchetii.Methods: We mainly investigated the anti-tumour activity of the physalins extracted from Physalis alkekengi var. franchetii on both solid and haematologic cancers. The main cells used in this study were NCI-H1975 and U266 cells. The major assays used were the CCK8 assay, western blot analyses, an immunofluorescence assay, the Annexin V assay, and xenograft mouse model.Results: The results showed that physalins exhibited strong anti-tumoural effect on both non-small cell lung cancer (NSCLC) and multiple myeloma (MM) cells by suppressing the constitutive STAT3 activity and further inhibiting the downstream target genes expression of STAT3 signalling, resulting in the enhanced apoptosis of tumour cells. Moreover, physalins significantly reduced tumour growth in xenograft models of lung cancer.Conclusions: Collectively, these findings demonstrated that the physalins from Physalis alkekengi var. franchetii may potentially act as chemopreventive or chemotherapeutic agents for NSCLC and MM by inhibiting the STAT3 signalling pathway. It is promising that the present study served as a guide to further explore the precise mechanism of Physalis alkekengi var. franchetii in cancer treatment.


Extraction
The extraction methods were previously described [13]. Eight-fold 95% EtOH under reflux was used to extract dried calyces (10 kg) of Physalis alkekengi var.
franchetii for three times. After vacuum drying, about 830 g of extract was obtained. The extract was resoluted in water (1 L) and then extracted three times with petroleum ether (1 L) and dichloromethane (1 L). The dichloromethane fraction (160 g) was obtained finally.

Quality control of Physalis alkekengi var. franchetii
Five physalins were separated and identified from the calyces of Physalis alkekengi var. franchetii in our previous study [13]. The extract contents of Physalis alkekengi var. franchetii were determinated by chromatographic analyses performed with an Acquity UPLC system (Waters, Milford, MS, USA) and a BEH C18 column (2.1 mm×100 mm×1.7μm). MS and MS-MS detections was done on a Micromass Quattro Premier tandem quadrupole mass spectrometer (Waters, Manchester, UK) using an electrospray (ESI) source in positive mode. The method was previously reported with minor modifications [13]. One hundred milligrams of pulverized sample was accurately weighed and was then transferred to a 50 mL volumetric flask and diluted with methanol to a volume of 50 mL. Finally, both of the test samples were filtered through a 0.45 μM membrane filter prior to UPLC analysis. The parameters of UPLC were as previously reported [13].

Cell viability
The tumour cell growth was quantified by the Cell Counting Kit-8 (CCK-8; DOJINDO) assay according to the manufacturer's instructions [14]. Briefly, the cells were plated in a 96-well plate (0.6 × 10 5 cells/well) and incubated with 100 μL of medium overnight. The cells were treated with different concentrations of physalins (0, 2.5, 5, 10, 15, 20, 30μg/mL) for 24h or 48h. The absorbance at 450 nm using a microplate spectrophotometer (Varioskan Flash, Thermo Fisher Scientific) after culturing the cells with 10 μL of CCK-8 reagent for about 2 hours. The IC50 value was used to evaluate the cytotoxicity of a drug which is needed for 50% growth inhibition in vitro. The IC50 value was calculated by the fitted line (Y = a× X + b).

Western blot analysis
Cold lysis buffer (150 mM NaCl, 1% NP-40, 50 mM Tris-HCL, pH 8.0, supplemented with complete set of protease inhibitors; Roche) was added to the treated NCI-H1975 and U266 cells. After centrifugation and concentration qualification, the whole protein extracts were mixed with loading buffer and then boiled for 10 min. After separating by SDS-PAGE, the proteins were transferred to PVDF membranes. 5% BSA was used for blocking. The primary antibodies were against STAT3, p-STAT3-Tyr, p-STAT3-Ser, Mcl-1, Bcl-2, Bcl-xL, survivin, cleaved caspased-3, cleaved caspase-9 and PARP (1:1000, Cell Signaling Technology, Beverly, MA, USA), β-Actin (1:3000 dilution, Sigma-Aldrich, Merck KGaA, St Louis, MO, USA). Membranes were then incubated with secondary antibodies (1:8000 dilution, Lianke Bio, P.R. China). The immunoreactive proteins were detected using a chemiluminescent immunodetection system (ChemiDocTM XRS). The semi-quantification of protein levels were performed by Image J software. The quantifications were shown by the relative gray values, as a ratio of each protein band relative to the lane's loading control. All the values were then analyzed by GraphPad Prism5 and shown in bars. * for P<0.05, ** for P<0.01 and *** for P< 0.001.

Apoptosis detection assay
The different cells (1×10 5 per well) mentioned above were seeded overnight on 12-well plates until 70% confluence, and then was treated with various concentrations of physalins (0, 5, 10, and 15μg/mL) for 24 h, as the control group was treated with DMSO. After trypsinizing, the cells were washed twice with cold PBS and were then mixed with binding buffer with anti-Annexin V-FITC/PI for 15 min according to the manufacturer's instructions. Then the apoptotic cells were evaluated using a FACS CantoII flow cytometer (BD Pharmingen) and the FlowJo software (TreeStarInc).

Immunofluorescence
The H1975 cells are treated with 15μM physalins for 6h followed by treating with recombinated IL-6 proetin (25 ng/mL) to induce pYSTAT3 levels and were then fixed in 4% paraformaldehyde for 15 min on ice and were permeabilized with pre-cooling methanol for another 10 min on ice. Next, the cells were blocked with blocking solution (5% normal goat serum, 0.3% Triton X-100 in PBS) for 60 mins at room temperature. After aspirating the blocking solution, the diluted primary antibody p-STAT3-Tyr (1:500, Cell signalling Technology) was applied and was incubated overnight at 4℃. The next day, the slides were rinsed in PBS 3 times and incubated with a fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer (1% BSA and 0.3% Triton X-100 in PBS) for 1-2 h away from the light. The slides were rinsed with PBS 3 times and coverslipped with Prolong® Gold Anti-Fade Reagent (#9071; Cell Signaling) with DAPI (#8961; Cell Signaling).

Xenograft mouse model
A total of 20 male BALB/c mice were divided into 4 groups (5 mice/group) and kept under pathogen-free conditions at room temperature (21 to 25 °C), with exposure to light for 12 h and 12 h of dark. Food and water were offered ad libitum. 5 × 10 6 human NCI-H1975 cells were subcutaneously injected into the right front leg of 6-weeks-old male BALB/c mice. When the tumour reached 100-150 mm 3 , 100 or 200 mg/kg/day (calculated according to clinical condition and converted using formula, i.e., Mouse dosage = X mg/kg×70 kg×0.0026/20 g ＝ 9.1 ×X mg/kg), physalins were administered orally with CMC-Na or 5 mg/kg/day cisplatin intraperitoneally injected for 12 days. The tumour volume and body weight were assessed every two days. The tumour volume was calculated as V = length × width 2 /2 [15]. The mice were sacrificed by cervical dislocation in a very short time, and then the tumours were obtaine and measured. For an animal carring one tumour, the diameter should not exceed 2.0 cm in mice for therapeutic studies according to the guidelines from the University of Pennsylvania Institutional Animal Care and Use Committee.

Statistical analysis
The statistical calculation of differences between the means was conducted by a Student's t-tests and shown as mean ± SD in each group. An analysis of variance (ANOVA), including the F value, was carried out with a Tukey's post hoc test for multiple comparisons. Statistical significance was considered at P<0.05 or P<0.01. All the experiments were replicated for three times.

Quality evaluation of Physalis alkekengi var. franchetii
According to previous studies, physalins are identified as the main active and characteristic component of Physalis alkekengi var. franchetii [9]. Five physalins were isolated and identified from the calyces of P. alkekengi var. franchetii in our previous study [13]. The results from UPLC-MS also showed that the main extracts contain five physalins, including physalin A, physalin G, physalin O, physalin L, and isophysalin, of which the contents were 26.03%, 2.66%, 52.06%, 12.92%, and 1.33% respectively (Fig. 1). However, based on a peak area normalization method, other components only occupied 5% in the extracts, and the extracts used in this study were endotoxin free. Thus, the effects of the other components on the in vitro experiment can be ignored.

Physalis alkekengi var. franchetii extracts inhibit cell viability in NCI-H1975 and U266 cells
To evaluate the anti-tumoural activities of physalins isolated from Physalis alkekengi var. franchetii, seven human tumour cells lines (NCI-H1975, NCI-H292, NCI-H358, U266, MKN45, MCF-7, and SW620) were treated with different dosages of extracts for 24 h or 48 h and were then subjected to a CCK8 assay (Cell Counting Kit-8, DOJINDO) ( Fig. 2A and B). The results showed that all the detected cell lines were sensitive to physalins, and the 50% inhibitory concentration (IC50) values of 24 h incubation were all less than 23 μg/mL. In particular, physalins had a higher inhibitory effect on NCI-H1975 and U266 cells (14.342 and 11.2 μg/mL, Table 1) and the IC50 values of these two cell lines had significant differences among other cells.
Previous studies show that STAT3 is activated and is highly expressed in these two cell lines, which is reminiscent of a connection between the inhibitory mechanism of physalins and the STAT3 signalling pathway.

Physalis alkekengi var. franchetii extracts reduce the phosphorylation (Tyr705) of STAT3 in NCI-H1975 and U266 cells
To test the above hypothesis, we initially observed the activity change in STAT3 after physalins treatment by examining the phosphorylation levels of STAT3 (Tyr705 and Ser727). NCI-H1975 and U266 cells were first administered with different concentrations of physalins treated for 4 h. The Western blot results showed that the level of STAT3 Tyr705 phosphorylation in the physalins-treated group decreased significantly, in a concentration-dependent manner, compared with the DMSO sovent group (Fig. 3A, B). By contrast, there is no obvious difference at the Ser727 site among all the groups. Moreover, the level of total STAT3 also showed no change in both cell lines. Collectively, these results suggested that physalins suppress constitutive STAT3 Tyr705 phosphorylation in NCI-H1975 and U266 cells.
Previous reports identified that STAT3 phosphorylation at Tyr705 (pYSTAT3) leads to STAT3's nuclear translocation in response to various extracellular cytokines, such as interleukin (IL)-6 [16]. So we examined whether physalins could repress the nuclear translocation of pYSTAT3 in NCI-H1975 cells. Immunofluorescence assay showed that the immunoreactivity of anti-pYSTAT3 was predominantly increased in the nucleus of IL-6-stimulated or unstimulated NCI-H1975 cells but was apparently decreased by a 15μg/ml physalins treatment for 6h (Fig. 3C).

Physalis alkekengi var. franchetii extracts suppress the expression of STAT3-regulated genes in NCI-H1975 and U266 cells
To further investigate whether physalins suppress STAT3-mediated downstream signalling, we detected the expression levels of target genes of STAT3, including anti-apoptotic proteins in the Bcl-2 family (Mcl-1, Bcl-2, and Bcl-xL), XIAP, and survivin by western blot analysis. The expression levels of Bcl-2 and XIAP in the NCI-H1975 and U266 cells were significantly reduced after the physalins exposure for 24 h. Moreover, the decrease occurred in a concentration-dependent manner (Fig.  4A，B). The band signals of both Bcl-2 and XIAP were strongly weakened after an incubation with physalins at 15 μg/mL. However, the expression levels of the other detected genes (Mcl-1, Bcl-xL and survivin) showed no significant difference. Therefore, these results indicated that P. alkekengi var. franchetii extracts further inhibited the STAT3-regulated downstream genes Bcl-2 and XIAP.

Physalis alkekengi var. franchetii extracts induce apoptosis in NCI-H1975 and U266 cells
Since Bcl-2 and XIAP are known to be the potent inhibitor of apoptosis, an Annexin V-FITC/PI assay was subsequently performed to investigate whether physalins further induce apoptosis in NCI-H1975 and U266 cells. As expected, the flow cytometry results showed that the physalins, in a concentration-dependent manner, significantly elevated apoptotic rate in the NCI-H1975 and U266 cells (Fig.  5A, B). Compared with the control groups, the proportion of apoptotic NCI-H1975 cells (upper right panel for the late stage plus the lower right panel for the early stage of apoptosis in Fig. 5A) increased from 6.8±1.2% to 11.5±3.1%~58.3±6.1% after physalins treatment at doses of 5~15 μg/mL for 24 h. Similarly, the percentage of apoptotic U266 cells in the physalins-treated groups (18.6±3.6%~43.4±10.4%) was much higher than the control group (4.5±1.0%). Correspondingly, the apoptosis related proteins cleaved caspase 3, cleaved caspase 9 and cleaved poly (ADP-ribose) polymerase (PARP) were significantly increased in the physalins-administered groups (24 h treatment) (Fig. 5C, D). Collectively, the above results demonstrated that P. alkekengi var. franchetii extracts promoted apoptosis in the NCI-H1975 and U266 cells by regulating the STAT3 signalling pathway.

Physalis alkekengi var. franchetii extracts inhibit tumour growth in a human NSCLC xenograft model
Finally, NCI-H1975 xenograft models were used to evaluate the anti-tumour activity of physalins. Comparing to the control group, the tumour growth (tumour volume) was significantly inhibited in the high-dose physalins-treated group (200 mg/kg) and the cisplatin (DDP)-treated (5 mg/kg) group under the surveillance of 12 days (Fig. 6A, B). Then, the mice were sacrificed, showing that the tumour weight was reduced in the low-dose physalins-treated group (100 mg/kg) (P=0.0016) and was obviously decreased in the high-dose physalins treated group (P<0.0001) and the DDP-treated group (P=0.0003) (Fig. 6C). The weights of the physalins-treated groups displayed no significant difference compared with the control group, but the DDP-treated group showed weight loss (P=0.01) (Fig. 6D). Taken together, the above results demonstrated that the anti-tumour activity of physalins was comparable to that of cisplatin, a chemotherapy agent, but physalins mitigate the side effect of body weight loss.

Discussion
Natural compounds derived from food, herbs and plants have been used as anti-tumour agents [17]. Population studies suggest that potential natural cancer preventive compounds are safe, practical, economical, and effective for cancer treatment [18]. For example, P. alkekengi var. franchetii is an herb that possesses popular interest due to its therapeutic properties [1]. Previous studies already demonstrate that several extractions from the calyces of P. alkekengi var. franchetii were vital in combating cancer [4,6]. For example, physalin A in different cencers [4,19]. It inhibits tumour cell growth, promotes apoptosis and autophagy via Nrf2 signalling, JAK-STAT3 signalling or ROS generation [4,20,21].
In our study, the main components from the calyces of P. alkekengi var.
franchetii extracts were five physalins, which were identified by UPLC. Physalins species are considered of great medicinal value, since these compounds display various of biological activities, such as antimicrobial, anti-tumour, anti-inflammatory, immunomodulatory, immunosuppressive, cytotoxic, trypanocidal, and molluscicidal effects [7,[22][23][24][25][26][27]. Previously, we have reported that anti-tumour growth and apoptosis-promoting effects of physalin A was depended on suppressing STAT3 activity in NSCLC cell lines [4]. Herein, our current study focused on the anti-tumour effect of the combination of five physalins from P. alkekengi var. franchetii. This antitumous effect was initially evaluated in seven types of cell lines and the results showed that physalins inhibited the growth of all the tested cell lines, which suggested that physalins exhibited a non-selective anti-tumour activity. Moreover, the anti-tumour molecular mechanism of physalins was explored in vitro using representative solid tumour (NCI-H1975) and haematological tumour (U266) cell lines. STAT3 is vital in the initiation and progression of vavious cancers, such as proliferation, anti-apoptosis, invasion and immune surveillance evasion [28][29][30]. It is activated by the phosphorylation of its tyrosine or serine residues via modulating upstream regulators [31,32]. The phosphorylation of STAT3 induces the dimerization through the interactions of reciprocal phosphotyrosine-Src homology domain 2 (SH2). Activated STAT3 dimers initiate the transcription of target genes by translocating to the nucleus and interacting with the promoter sequence [31,33]. Parent STAT3 activation is related to a wide spectrum of cancers, including NSCLC and MM, and is positively associated with a poor prognosis [34][35][36][37]. A wide range of pre-clinical studies show that phosphorylated-STAT3 (pSTAT3) is a well-known characteristic of various cancers [38][39][40]. Accumulating evidence shows that the constitutive STAT3 activation occurs frequently in a wide range of tumour cells, including MM and more than 22%~65% of NSCLC [41][42][43][44]. This indicates that the aberrant STAT3 signalling is an important process in malignant progression. As such, the inhibition of STAT3 signalling is considered a novel and potential target to treat cancers. STAT3 is activated by Tyr705 site phosphorylation in response to membrane factors such as the interleukin-6 (IL-6) [45]. It also has been shown that phosphorylation of Ser727site of STAT3 is phosphorylated by various kinds of kinases in various cancers [46][47][48][49]. However, some reports have showed that the Ser727 site phosphorylation suppresses STAT3 activity and cell proliferation [50,51]. In our study, we reported that P. alkekengi var. franchetii extracts repressed Tyr705 phosphorylation of STAT3 in H1975 and U266 cells, but have no significant effect on Ser727 phosphorylation of STAT3.
In our previous study, we illustrated that physalins acted as Michael reaction factors and directly induced a rapid drop in the concentration of intracellular glutathione (GSH), thereby triggering the S-glutathionylation of STAT3 and inhibiting the tyrosine phosphorylation of STAT3 [13,52]. In this study, P. alkekengi var.
franchetii extracts inhibited the level of STAT3 tyrosine phosphorylation and further suppressed the expression of the STAT3 downstream target genes. Bcl-2 and XIAP are both anti-apoptotic proteins. Following Bcl-2 downregulated, the pro-apoptotic proteins are subsequently activated, resulting in mitochondrial outer membrane permeabilization (MOMP). Then cytochrome c and second mitochondria-derived activator of caspase (SMAC) released from the mitochondria, which results in the activation of caspase 9. Caspase 9 then activates procaspase 3 and procaspase 7, leading to cell death [53]. XIAP, X-linked inhibitor of apoptosis, inhibits caspase 9 [54]. Another apoptotic marker cleaved PARP is catalyzed by caspase-3 [55]. In brief, P. alkekengi var. franchetii extracts inhibit STAT3-targeted anti-apoptotic proteins, which leads to the cleavage of both caspase-3 and PARP, resulting in an increased apoptosis rate in both NCI-H1975 and U266 cells. Therefore, suppressing the continuous activation of STAT3 may be an effective target for cancer therapy. It is meaningful and valuable to discover and develop effective STAT3 inhibitors as an anti-tumour component in the treatment of cancer. However, the inhibitory specificity and selectivity of physalins are still unknown, which will be further clarified by a molecule docking assay and competitive inhibition experiment.
Notably, based on the results from the in vitro experiments, including cell proliferation and apoptosis, we are aware that the effects of P. alkekengi var.
franchetii extracts on NCI-H1975 and U266 cells are almost consistent, which suggests that physalins from P. alkekengi var. franchetii may have similar or identical anti-tumour activity on solid tumours as well as haematological tumours. Moreover, the anti-tumour effects of the P. alkekengi var. franchetii extracts were tested using a human xenograft model of lung cancer. Consistent with the in vitro results, the data showed that physalins from P. alkekengi var. franchetii inhibited tumour growth, which was embodied in a decreased tumour volume and weight in the xenograft model.
In a human NSCLC xenograft model, the body weights of the physalins-treated groups were no significantly different compared with the control group, but DDP-treated group showed weight loss. Moreover, the physalins-treated groups did not have an adverse reaction during observation, and thus, P. alkekengi var. franchetii extracts are safe, have a low-toxicity and no obvious side effect. Compared with the effect of Physalin A (injected intraperitoneally ) [4], Physalis alkekengi L. extracts (taken orally) are easier to take than Physalin A only. P. alkekengi L. extracts are more convenient for large-scale preparation and extraction than the isolated and purified physalin monomer. Moreover, an herbal cocktail therapy can partially reduce single drug resistance. The anti-tumor effects of other physalins and their combined inhibitory effects should be further discovered in our next paper In summary, the P. alkekengi var. franchetii extracts inhibited tumour proliferation and promoted apoptosis in NSCLC and MM cells by repressing the STAT3 signalling. The findings of this study help the development of physalins from P. alkekengi var. franchetii as a promising anti-tumour drug. Clinical evaluation and application were the main limitations of our study, and these can be the future direction.   The cell lysates were subjected to a Western blot analysis using antibodies specific for p-STAT3-Tyr, p-STAT3-Ser, STAT3 and β-actin. The semi-quantification of protein levels were performed by Image J software. The relative gray values of p-STAT3-Tyr were shown below. (C) The physalins suppressed p-STAT3 nuclear translocation. H1975 cells were treated with 15μg/ml of physalins for 6h with or without 25 ng/mL of IL-6. The immunofluorescence analysis was performed with a p-STAT3-Tyr primary antibody followed with an anti-rabbit IgG Fab 2Alexa Fluor 555 antibody. Coverslipped slides were covered with Anti-fade reagents with DAPI. The merged images show the overlay of the red Alexa Fluor 555 and the blue DAPI fluorescence.