Sijunzi Tang Enhances The Sensitivity of Lung Cancer Cells To Getinib Based On Glutamine Metabolism

Background: Acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) has been the major bottleneck that limits the long-term clinical ecacy. Therefore, the exploration of novel strategies for the treatment of non-small cell lung cancer (NSCLC) is urgent. Sijunzi Tang (SJZ) has been usually used as a complementary therapy for cancer patients that can prolong their overall life. Nevertheless, its underlying mechanism when combined with EGFR-TKIs is not clear. Methods: Here, the anti-tumor activity was evaluated by determining cell viability by using the Cell Counting Kit-8 (CCK-8). A high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was established to simultaneously quantify nine metabolites in the glutamine metabolic pathway, such as glutamine, glutamate, αketoglutaric acid, fumarate, succinate, citrate, cis-aconitate, alanine, and malate in PC-9/GR cells. Results: The results showed that SJZ could enhance the sensitivity of lung cancer cells to getinib. The content of glutamine and glutamate in the SJZ/getinib group was signicantly lower than that in the getinib group, while the concentration of other metabolites did not change signicantly. Conclusion: In summary, SJZ can reverse getinib resistance by modulating glutamine metabolism. Our results suggested that SJZ might be a potential resistance reversal herbal medicine and combined with getinib might be a promising strategy for the therapy of lung cancer. Also, the research will provide novel insights for the study of the mechanisms on Traditional Chinese medicine (TCM) prescriptions-reversed resistance of EGFR-TKIs, and scientic basis for the combined treatment of lung cancer with TCM and EGFR-TKIs. SJZ/getinib by HPLC-MS/MS technology. Changes in the content of related metabolites in the glutamine metabolic pathway. Based on this, we measured the content changes of 9 metabolites including glutamine. Related metabolites in the glutamine metabolic pathway are polar compounds, which have small reservations on the conventional column. We found that waters ACQUITY HSS T3 column (2.1×100 mm, 1.8μm) could achieve satisfactory separation, which is reliable in terms of determining the metabolites eciently. Our research results show that the content of glutamine and glutamate in the combined administration group was signicantly lower than getinib group, but there was no signicant change compared with the model group; and when glutamine was converted to αketoglutaric acid after entering the tricarboxylic acid (TCA) cycle, the content of fumarate, succinate, citrate, malate, cis-aconitate, and alanine did not change signicantly among the administration groups, which suggests that SJZ is through down-regulation of glutamine and glutamate content to reverse getinib resistance. Glutamine is a non-essential amino acid. Studies have shown that the expression level of glutamine in lung cancer tissues increases, especially in NSCLC 16 . Glutamine makes many


Introduction
Lung cancer ranks as a leading cause of cancer-related deaths worldwide in the light of high mortality and poor survival, and most patients (84%) suffer from non-small cell lung cancer (NSCLC) 1,2 . The current practices recommend ge tinib as rst line treatment to advanced NSCLC patients with an activated epidermal growth factor receptor (EGFR) mutation. Ge tinib, one of the rst-generation EGFR tyrosine kinase inhibitors (TKIs), selectively and reversibly blocks the intracellular EGFR signaling pathway to suppress the growth, metastasis, and angiogenesis of tumors. Initially, the unprecedented survival bene ts of ge tinib are impressive. While most of the patients treated with ge tinib ultimately develop acquired resistance after 9 to 14 months of treatment, rendering the overall survival of patients unsatisfactory 3 . The inevitable barrier limits the effectiveness of ge tinib and remains a pervasive challenge for long term treatment. For this reason, it is necessary for the development of new treatment strategies to overcome ge tinib resistance.
As a highly heterogeneous disease, NSCLC has many patterns of gene expression that are not dependent on a single signaling pathway. Several molecular mechanisms of ge tinib resistance have been identi ed in the past few decades, including secondary mutation of EGFR (T790M mutation), mesenchymal-epithelial transition factor (MET) ampli cation, hepatocyte growth factor (HGF) overexpression, and genetic alterations 4 . However, in up to approximately 30% of cases, the mechanisms are still unexplained. It has been reported that glutamine metabolism is related to many oncogenes and tumor suppressors [5][6][7] . As con rmed in an emerging study, ge tinib is a potent inducer of cancer cell death by inhibiting the utilization of glutamine and reducing the production of Adenosine triphosphate (ATP) and glutathione (GSH) in PC-9 cells, but ge tinib has no effect on the production of ATP and GSH in ge tinibresistant PC-9 cells, which even can use glutamine for proliferation 8 . The researchers speculated that restricting glutamine metabolism might be a potential strategy to delay ge tinib resistance.
In recent years, Chinese medicine has been considered as an alternative and complementary therapy in cancer treatment with approved curative effects, which effectively delay progression, extend survival, and reduce adverse reactions in cancer patients [9][10][11] . Si Jun Zi Tang (SJZ), a four-herb Chinese medicine formula, was rst recorded in the "Prescriptions People's Welfare Pharmacy". SJZ is obtained by decocting Panax ginseng C.A.Mey., Atractylodes macrocephala Koidz., Poria cocos (Schw.) Wolf and Nardostachys jatamansi DC in an appropriate ratio 1 . It has been reported that SJZ plays a crucial role in preventing tumor recurrence and metastasis, improving life quality, and prolonging the overall survival time 12 . In our previous study, it has been con rmed that SJZ signi cantly enhances the inhibitory effect of ge tinib on tumor growth and metastasis in the Lewis lung carcinoma (LLC) mice model 13 . In addition, our results suggest that SJZ might enhance the sensitivity of ge tinib via the regulation of corresponding metabolites. However, the speci c mechanism requires further elucidation.
Therefore, we propose that ge tinib resistance could be reversed by SJZ through the glutamine metabolism pathway. The cell viability was determined using the cell counting kit-8 (CCK-8) assay. A high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) method was established and validated to determine the alterations of metabolites in the glutamine metabolism pathway.

Chemicals and reagents
Methanol, acetonitrile, and formic acid (Fisher Scienti c, Loughborough, UK) were used HPLC-grade.

Cell viability assay
The inhibitory effects of ge tinib on cell proliferation were evaluated by CCK-8 (Dojindo, Kumamoto, Japan). PC-9/GR were cultured in 96-well plates at a density of 1 × 10 4 cells per well for 24h and then treated with ge tinib and/or SJZ at the indicated concentrations. After incubating for 48 h, the cells were washed twice with PBS and incubated with CCK-8 working solution for 1 h at 37°C, according to the manufacturer's protocol. Through iMark™ Microplate Absorbance Reader (Molecular Devices, Sunnyvale) measuring absorbance at 450 nm.

Standard stock solutions, calibration standards, and quality control samples
Glutamate, glutamine, alanine, cis-aconitate, α-ketoglutaric acid, fumarate, succinate, citrate, malate, and glu-d5 were dissolved in 0.1% formic acid in water to obtain a concentration of 5 mg/mL standard stock solutions. Putting stock solutions of all analytes and IS at -20°C for further study. The working solutions of four analytes such as glutamate, glutamine, alanine, and cis-aconitate were determined in ESI+ mode, and the other working solution of ve analytes such as α-ketoglutaric acid, fumarate, succinate, citrate, and malate were measured in ESI-mode. Stock solutions of the analytes were prepared by step wisely diluting with cell extraction solvent to generate the calibration standards at 6-12 different concentrations. For the investigation of precision and stability, quality control (QC) samples were prepared by using separate aliquot of the stock solution in accordance with the same way but independently from the calibrators. The levels of lower limits of quanti cation (LLOQ), and QCs were shown in Table 2. All standard and QC solutions contain and all measured samples contain internal standard (IS) (173.2 ng/mL). All of them were consisted of methanol-water (80:20, v/v).

Sample collection and preparation
PC-9/GR cells were seeded in a six-well culture plate (Corning) at a density of 5 × 10 5 cells/well. Harvesting the cells when the cells are 80-90% con uence, discarding the medium, and washing twice with cold PBS, then using liquid nitrogen quenched. Cells were lysed with cold extraction solvent and by using a cell scraper to harvest. Transfer the extract to a 1.5 mL test tube (from Eppendorf) and place it in a refrigerator at -80°C for later use. Centrifuge the mixture at 14,000 rpm (4°C, 15 minutes) to remove cell debris and collect the supernatant. Transfer the supernatant to a vial for analysis and aliquot and store it at -80°C. The content of each analyte is quanti ed by the protein concentration of the cell pellet.

Therapeutic drug treatments
After culturing the PC-9/GR cells for 24 hours, they were cultured in a medium containing Sijunzi tang, Ge tinib, Sijunzi tang/ge tinib and complete medium according to the aforementioned conditions for 48 hours, respectively. Then the cells are processed according to the previously described steps.

Method validation
The calibration curve of the analyte is obtained by plotting the relationship between the peak area ratio of each analyte to IS and the concentration of the analyte. Linearity is measured by the correlation coe cient (r). Using MultiQuant™ software (version 3.0.3) to integrate the peak area. Theoretically, the requirement of limit of detection (LOD) is 3. LOQ is de ned as a S/N ratio of 10. LLOQ is the lowest concentration of an analyte that can be accurately quanti ed in a sample. Since the 9 analytes are endogenous, cell lysates are used as a biological matrix. The accuracy of QC samples is expressed as relative standard deviation (RSD). The stability is tested by evaluating the concentration change of analytes in the QC sample after preparation. The prepared QC sample was tested ve times at room temperature for 12 hours, and then analyzed.

Statistical analysis
The experimental results were analyzed using Graphpad Primer 8.0 statistical software. One-way analysis of variance (one-way ANOVA) was used for the comparison of measurement data among multiple groups, and the t-test was used for comparison between the two groups. When P<0.05 indicates a signi cant difference, and P<0.01 indicates a very signi cant difference.

SJZ enhanced the sensitivity of lung cancer cells to ge tinib
To investigate the effect of SJZ on reversing ge tinib resistance, choosing PC-9/GR cells as representative ge tinib-resistant lung cancer cells. After PC-9/GR cells were pretreated 48h with SJZ or ge tinib or a combination of them, the viability of cell lines was evaluated using CCK-8. Ge tinib and SJZ inhibited tumor cell growth in a dose-dependent manner as shown in Figure 1A and B. The IC 50 values of ge tinib and SJZ on PC-9/GR cells were 4.81µg/ml mL and 7.81mg/mL, respectively. When the concentration of ge tinib was lower than 0.780µg/mL, more than 90% of the cell viability was not affected. Thus, three concentrations, 0.0159, 0.112, and 0.780 µg/mL were used in the following experiments. Subsequently, as 1.62 mg/mL SJZ combined with the above three concentrations of ge tinib, SJZ signi cantly enhanced the inhibitory effects of ge tinib on cell proliferation (Supplementary Figure S1). Furthermore, the results of JinZhengJun Q value analysis showed that 0.0159 µg/mL ge tinib plus 1.62 mg/mL SJZ had a synergistic inhibitory effect because Q value was 1.17. Q value was calculated using the JinZhengjun Method to evaluate the combination e cacy, Q >1.15, named as synergy, Q =0.85~1.15, named as an additive effect, and Q <0.85, named as antagonism effect 14 . Interestingly, when the SJZ concentration was reduced to 1.089 mg/mL combined with ge tinib, 0.0159 and 0.112 µg/mL ge tinib had a synergistic inhibitory effect except for 0.780 µg/mL, and the Q values were 1.26 and 1.29 ( Figure 1C and D). In particular, we continued to reduce the concentration of SJZ to 0.0159 mg/mL in the next experiment, which did not affect more than 90% of cell viability. The results demonstrated that 0.0159 and 0.112 µg/mL ge tinib had a synergistic inhibitory effect except for 0.780 µg/mL (Supplementary Figure S2).

Optimization of chromatography and mass spectrometry conditions
Methanol and acetonitrile are widely used in chromatographic separations. In our experiments, acetonitrile provided higher selectivity and elution strengths than methanol in the light of chromatographic retention mechanisms and behaviors. 5 mM ammonium acetate resulted in a signi cant decrease in the analyte response and high ionic strength might lead to poor ESI performance and ion-ion suppression. Therefore, 0.1% formic acid was selected to improve ionization e ciency and optimize the ow rate and gradient elution to ensure symmetrical peak shapes. Each analyte and IS was rst infused into the mass spectrometer and their molecule were used for method development. Using transitions to check the sensitivity and speci city of each analyte in biological samples. The highest sensitivity of fumarate, succinate, citrate, malate, α-ketoglutaric acid was achieved in ESI-mode, while glutamate, glutamine, alanine, cis-aconitate, and IS were acquired in ESI+ mode. Moreover, the fewer MRM transitions were detected at one time, the higher intensity of each MRM transition. The typical MRM chromatograms of 9 metabolites and IS are shown in Figure 2. All compounds were determined and analyzed to obtain speci c and sensitive MRM transition ion pairs. In addition, CE and DP conditions were optimized to ensure each analyte had the highest sensitivity. Stable isotopelabeled IS(SIL-IS) was used to avoid ionization inhibition or enhancement of analytes in the complex biological matrix by ESI. The selected ion pairs and the optimal parameters for analyte detection (identi ed by Analyst® Software Version 1.6.3) are shown in Table 1.

Methods validation
The linearity, correlation coe cient, LLOQ, and LQC of quantitative compounds are shown in Table 2 and Table 3 respectively. The results showed that within a certain linear range, the correlation coe cients are all above 0.99, which means that the concentration has a good linear relationship with the peak area. The LLOQ was in the range of 8.5-4065ng/ml.  3.4 Application to the study of glutamine metabolism in lung cancer cells The altered cellular metabolism was recently reconsidered as a core hallmark of cancer, which is also closely associated with drug resistance. The method has been developed to quantify the metabolites related to glutamine metabolism pathway in PC9/GR cells. The concentration changes of 9 metabolites were summarized in Figure 3. Compared with the model group and SJZ group, the concentration of glutamine and glutamate in PC-9/GR cells decreased in the SJZ combined with the ge tinib group, however, alanine and cis-aconitic acid had no signi cant change. Additionally, fumarate, succinate, citrate, α-ketoglutaric acid had no signi cant change among the groups. Compared with the model group, the concentration of malate was lower in the combined with ge tinib group.

Discussion
The experimental results reveal that SJZ can increase the sensitivity of PC-9/GR to ge tinib. Different concentrations of SJZ, the inhibition rate of PC-9/GR varies between 5.55-26.17%, combined with the resistance concentration of ge tinib, had a synergistic inhibitory effect on PC-9/GR because the CI values are all lower than 0.9. Moreover, as the concentration of SJZ decreased, the synergistic effect was stronger. The results indicated that the synergistic effect was the strongest under the condition that neither SJZ nor ge tinib affected the viability of more than 90% of cells.
A recent study showed that ge tinib resistance is mainly caused by glutamine related metabolic pathways when compared with ge tinib-sensitive cells 15 . Further studies have shown that ge tinib inhibits the utilization of glutamine in ge tinib sensitive cells and reduces the production of ATP and GSH, thereby inducing cancer cell death. However, ge tinib has no effect on the ATP and GSH of drugresistant cells, and drug-resistant cells can use glutamine for proliferation. To further clarify how SJZ reverses ge tinib resistance, we measured PC-9/GR cells after administering SJZ, ge tinib, and SJZ/ge tinib by HPLC-MS/MS technology. Changes in the content of related metabolites in the glutamine metabolic pathway. Based on this, we measured the content changes of 9 metabolites including glutamine. Related metabolites in the glutamine metabolic pathway are polar compounds, which have small reservations on the conventional column. We found that waters ACQUITY HSS T3 column (2.1×100 mm, 1.8μm) could achieve satisfactory separation, which is reliable in terms of determining the metabolites e ciently. Our research results show that the content of glutamine and glutamate in the combined administration group was signi cantly lower than ge tinib group, but there was no signi cant change compared with the model group; and when glutamine was converted to αketoglutaric acid after entering the tricarboxylic acid (TCA) cycle, the content of fumarate, succinate, citrate, malate, cis-aconitate, and alanine did not change signi cantly among the administration groups, which suggests that SJZ is through down-regulation of glutamine and glutamate content to reverse ge tinib resistance. Glutamine is a non-essential amino acid. Studies have shown that the expression level of glutamine in lung cancer tissues increases, especially in NSCLC 16 . Glutamine makes many contributions in redox homeostasis, mating energy, macromolecule synthesis, and cancer cell signaling 17 . In glioblastoma cells, increased glutamate levels can provide a fuel cycle for TCA 18 . Glutamine is catabolized by GLS1 to produce glutamate, and then glutamate is produced by the action of glutamate dehydrogenase or glutamate related aminotransferases, such as alanine aminotransferase and aspartate aminotransferase. Glutamate is converted to α-ketoglutaric acid and NH4+, αketoglutaric acid provides a carbon source for the synthesis of various biological precursors in the TCA cycle, which are essential for the survival and proliferation of tumor cells. Although cancer cells performed obvious Warburg effect, they can still maintain a complete TCA cycle and gradually become more dependent on glutamine metabolism, which makes it possible for cancer cells to use TCA cycle intermediates as a precursor to biosynthetic pathway 19,20 . Most of the α-nitrogen produced by the degradation of glutamine is secreted from the cell in the form of ammonia and alanine, and the α-nitrogen will be mainly made use of maintaining the intracellular amino acid pool. The results of the study indicate that the amino group derived from glutamate secretion is necessary for the use of glutamine as a precursor for angioplasty and the production of NADPH 19 . By stimulating the decomposition of glutamine, transforming cancer cells become glutamine addiction to maintain strong cell proliferation., A higher ratio of glutamate to glutamine is associated with prolonging overall survival in breast cancer patients 21 . The enhanced glutamine metabolism pathway helps to increase the survival rate of cancer cells. Compared with the loss of glutamine catabolism ability, ge tinib-resistant cells still make use of glutamine as a response to ge tinib. In addition when circulating glutamine levels were reduced, VM-M3 cells proliferation was signi cantly inhibited and long-distance transfer in vitro and in vivo was also inhibited 22 . In short, the reduction of glutamine level helps to inhibit cell growth, which also shows that SJZ can reverse the resistance of ge tinib by reducing the concentration of glutamine and increase the resistance of ge tinibresistant cells to ge tinib. Taken together, our research shows that SJZ can reverse ge tinib resistance by reducing the concentration of glutamine and glutamate. SJZ regulates the changes in the content of glutamine and glutamate in the glutamine metabolism pathway. The recognition that alterations in glutamine metabolism plays a signi cant role in the development and progression of cancer has driven continuous efforts to exploit this metabolic changes to treat cancer 23 . However, how SJZ regulates the targets on the glutamine metabolism pathway to affect the content of glutamine and glutamate, thereby reversing ge tinib resistance, is still unclear. Therefore, the mechanism of how SJZ regulates the pathway of glutamine metabolism needs further study. This study will provide some inspiration for the treatment of ge tinib resistance in NSCLC patients, as well as strategies to reverse the resistance of targeted drugs.

Conclusion
we found that SJZ can enhance the sensitivity of lung cancer cells to ge tinib. The content of glutamine and glutamate in the SJZ/ge tinib group was signi cantly lower than that in the ge tinib group, while the concentration of other metabolites did not change signi cantly. Therefore, SJZ can reverse ge tinib resistance by modulating glutamine metabolism. Our ndings will provide novel insights for the mechanisms study on Traditional Chinese medicine (TCM) prescriptions-reversed resistance of EGFR-

Availability of data and materials
All the data used to support the ndings of this study are available from the corresponding author upon reasonable request.
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Consent for publication
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Competing interests Figure 1 Ge tinib(A) and SJZ(B) inhibit cell proliferation in PC-9/GR cells. PC-9/GR cells were treated with SJZ and/or ge tinib at the indicated concentrations for 48 h. Cell inhibition was measured by CCK-8, and IC50 values were calculated (C). The concentration of SJZ was 1.089 mg/mL, but the concentration of ge tinib was indicated respectively (D). JinZhengJun Q value of the combination of SJZ and ge tinib.  Representative chromatograms of a standard sample containing glutamate, glutamine, cis-aconitate, alanine respectively, and internal standard (IS) (A). Extracted ion chromatograms of IS, Glu-d5 (B).