*2.5. Lactucin Inhibits A549 Cell Proliferation by Downregulating MAPK/ERK and Central Carbon Metabolism Pathways*

Natural Lactucin was conjugated with Propargylamine through Michael's addition reaction. Structure of resulting Lactucin-Propargylamine conjugate [(3aR,4S,9aS,9bR)-4 hydroxy-9-(hydroxymethyl)-6-methyl-3-((prop-2-yn-1-ylamino)methyl)-3,3a,4,5,9a,9bhexahydroazuleno[4,5-b]furan-2,7-dione] was confirmed using 1H Nuclear Magnetic Resonance (NMR), Figure 5A. The yield of the probe was estimated at 50% using High Performance Liquid Chromatography (HPLC). Cytotoxicity of the Lactucin probe and Propargylamine showed that the Lactucin probe exerts similar antiproliferation activity on A549 cells

as natural Lactucin does both, doses dependent (Figure 5B) and time-dependent (Figure 5C) manners. In contrast, Propargylamine does not exert any significant bioactivity.

**Figure 4.** Lactucin inhibits A549 lung cancer cells by downregulating the MAPK/ERK pathway. (**A**) WB analysis of PTEN, Akt, p-Akt, MEK 1/2, p-MEK, ERK, and pERK 1/2 protein expression in A549 cell after exposure to Lactucin at 0, 20, 40, and 80 μM for 24 h. (**B**) Data are expressed as the means ± standard deviation of triplicate experiments. \* *p* < 0.05, \*\* *p*< 0.01, and \*\*\*\* *p* < 0.0001 indicate result's significant difference from the control.

During the Activity-Based Protein Profiling (ABPP) assay, after Co-Immunoprecipitation (Co-IP), isolated protein lysate resolved on the Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) showed five distinct bands in the treatment column after coomassie blue staining (Figure 6B). After testing these five bands using Liquid Chromatography with tandem Mass Spectrometry (LC-MS/MS) and analyzing them using MASCOT against 2019 human proteins. Many probable matches were found. Functional annotation clustering of LC-MS/MS results with the metabolic markers (upregulated and downregulated) data using DAVID Bioinformatics resources showed Lactucin interacts with several key enzymes of the "Central carbon metabolism in cancer" pathway (Kyoto Encyclopedia of Genes and Genomes, KEGG). These interacting proteins were identified as L01 = Phosphoglycerate mutase 1/2/4 (PGM, 70 kDa), L02 = Pyruvate kinase M1/2 (PKM, 58 kDa), L03 = Pyruvate dehydrogenase E1 subunit alpha 1/2 (PDH, 43 kDa), L04 = Lactate dehydrogenase A (LDHA, 37 kDa), but L05 was not identified. At the beginning of this

manuscript, we defined cancer and tumor as proliferation disorders. However, they often show increased glucose and glutamine consumption, lactate secretion rate, glycolysis rate, and modified use of metabolic enzyme isoforms [27]. KEGG "Central carbon metabolism in cancer" pathway (Supplementary Material Figure S1 and Figure 6C) shows the difference in carbon metabolism between normal and malignant cells. Lactucin binding to PGM and PKM lower pyruvate synthesis, which binds to LDHA to lower Lactucin synthesis. Lactucin also binds to PDH, limiting the availability of Acetyl CoA entering the Tricarboxylic Acid (TCA) cycle and ultimately reducing lactate production, a hallmark of a metastasized tumor. Reducing PGM, PKM, and LDHA overexpression is attributed to the upregulation of p53 in response to DNA damage. From the KEGG pathway for NSCLC (Supplementary Material Figure S2 and Figure 6C), we see several oncogenes, such as p53, p21, and Bax, were upregulated; Cyclin D1, Akt, MEK, ERK, and CDK4 were downregulated.

**Figure 5.** The structure of Lactucin-Propargylamine conjugate was confirmed using 1H NMR (**A**). Inset showing schematics of synthesis of Lactucin probe where reagents and conditions for synthesis were (a) propargylamine, pyridine, 0 ◦C, 36 h. Upon cytotoxicity evaluation on A549 cells, the Lactucin probe showed similar bioactivity as natural Lactucin compared to Propargylamine in incremental dose (**B**) and progressive dosing time (**C**) experiments.

**Figure 6.** Lactucin downregulates Raf-MEK-ERK by inhibiting upstream trans-membrane peptides. Biotinylated Lactucin probes bound to targeted proteins are immobilized to streptavidin agarose through biotin, azide, and a linker molecule (**A**). Visualizing SDS resolved A549 protein lysate showed biotinylated Lactucin probe binds five peptides (**B**). Lactucin downregulates A549 lung adenocarcinoma cells Raf-MEK-ERK pathway and Central Carbon Metabolism in Cancer cells (**C**).

#### **3. Discussion**

In the present experiment, we observed that Lactucin has significant antiproliferative, G0/G1 cell cycle arrest, and apoptosis-inducing properties on A549 and H2347 lung adenocarcinoma, not on MRC-5 normal lung cells. Firstly, Lactucin-induced time and dose-dependent inhibition of A549 and H2347 cells were observed, and then their IC50 values were determined to be 79.87 μM and 68.85 μM, respectively. According to WHO [28], for L-6 (rat skeletal myoblast) cells, compounds with an IC50 above 90 μM are not cytotoxic, IC50 between 2–80 μM is moderately cytotoxic, and IC50 below 2 μM is cytotoxic. For natural compounds, Ren, et al. [25] described an IC50 below 100 μM to be cytotoxic and reported Lactucin inhibits KB and Bel 7402 cells with an IC50 value of 75 μM and 55 μM, respectively. Lima, et al. [29] considered IC50 of <40 μg/mL and <4 μg/mL for plant extract and pure compounds respectively to be cytotoxic. According to American National Cancer Institute (NCI), except for fibroblast, an IC50 of ≤30 μg/mL of plant extract is cytotoxic, but they did not specify pure natural compounds. IC50 values we observed in adenocarcinomas can thus be considered cytotoxic. The LC3-II and LC3-II/LC3-I ratio increase is a classic sign of autophagy [30]. However, this is not always the case due to the lower sensitivity of LC3-I than LC3-II in WB and the higher degradation rate of LC3-II in the presence of lysosomal protease inhibitor [30,31]. We observed a dose-dependent increase of LC3-II in H2347 and LC3-I in A549 and H2347 cells. It was probably due to the concomitant increase in LC3 production and LC3I to LC3II conversion, rapid degradation of LC3-II, or lower detection of LC3-I in WB. In conclusion, LC3 WB results didn't sufficiently indicate that Lactucin directly induces autophagy. LC3-I and LC3-II increase was probably due to autophagosome increase by some other means [30].

We found Lactucin induces G0/G1 cell cycle arrests from cell cycle analysis. Specific cell cycle-related proteins, such as cyclins and CDKs, positively upregulate the cell cycle, whereas CDKs inhibitors stop the Cyclins and CDKs unit assembly [32]. In this experiment, we observed the downregulation of Cyclin B1, Cyclin D1, CDK2, and CDK4, and the upregulation of p21 and p53. Downregulation of Cyclin D1 and upregulation of p53 were associated with G0/G1 cell cycle arrest and apoptosis [32], which coincides with our findings. Lactucin-induced inhibition of cell proliferation was also reported by Zhang, et al. [24] in human HL-60 leukemia cancer cells and by Wang, et al. [22] in mouse 3T3-L1 fibroblast cells. Though in 3T3-L1 G0/G1 cell cycle arrest was reported, [22] in HL-60 cells, Sub-G1 cell cycle arrest was reported [24].

We also observed an increase in early and late apoptosis in A549 and H2347 cells induced by Lactucin. Lactucin induces apoptosis by upregulating the expression of mitochondrial apoptosis-related proteins, such as c-Caspase, c-PARP, and Bax, while downregulating the expression of Bcl-2. Zhang, et al. [24] reported Lactucin induces apoptosis in HL-60 cells by swelling up mitochondria and endoplasmic reticulum (ER) on the transition electron microscope (TEM). Jang, et al. [33] reported that Lactucin induces ROS-mediated apoptosis in human renal cancer cell Caki-1 by downregulating Bcl-2 expression and CFLARL stability where Bcl-2 downregulation was at a transcriptional level caused by inactivation of the NF-κB pathway. However, we observed concurrent upregulation of PTEN and downregulation of Akt, MEK, and ERK phosphorylation narrowed down the Lactucin-influenced NSCLC pathway to PI3K/Akt and MAPK/ERK. McCubrey, et al. [34] suggested that ERK can activate the NF-κB transcription factor (nuclear factor immunoglobulin κ chain enhancer-B cell) by phosphorylating and activating inhibitor κB kinase (IKK) through an indirect mechanism. We also performed an ABPP assay on A549 protein lysate using a Lactucin-Propargylamine probe. Structural activity relationship (SAR) studies by Ren, et al. [25] revealed that in SL, the position 8 ester group (γ-butyrolactone) and the methylene group at exocyclic position 11 (α), play a significant role in antitumor activities of Lactucin-like guaianolides (Figure 5A) [16]. α methylene γ lactone, the' enone' or unsaturated carbonyl system (O=C–C=CH2) was also reported to increase the toxicity towards tumor cells [15]. While synthesizing the Lactucin-Propargylamine probe, we avoided the 8-ester group and 11 methylene group positions mentioned earlier. However, adding the alkynyl group to 11 (α), exocyclic methylene didn't hinder its cytotoxicity against A549 cells (Figure 5B,C). It also suggests that Lactucin may simultaneously exert its activity through different groups. Proteins isolated using this probe were analyzed in LC-MS/MS and combined with WB results for functional enrichment. In the DAVID functional enrichment of western blot and ABPP peptides, four were identified as key enzymes in central carbon metabolism in the cancer cell (Figure 6C). Overexpressed and modified central carbon metabolism enzymes are essential to support the exponential growth of the metastasized tumor. Regulating their expression will limit the available energy and thus control or seize tumor growth.

In anticancer therapy, oncogenes and tumor suppressors are two likely targets for inhibiting cancer cells [26]. From the KEGG pathway of NSCLEC by Kanehisa Laboratories, we know that in the case of NSCLC like A549 or H2347, we know the potential oncogene and tumor suppressors targets. DAVID enrichment results showed that in the NSCLC, Lactucin downregulates MEK-ERK pathway, resulting in the downregulation of CyclinD1, CDKs, and upregulation of the p53. p53, in turn, upregulates p21 and Bax. It doesn't show interaction with any of the upstream components of NSCLC. When DAVID enrichment for the KEGG pathway "Central Carbon Metabolism in Cancer" was plotted, it showed four Lactucin interacting enzymes besides the western blot findings. Downregulation of MAPK/ERK observed in Figure 6C also lowers the expression of c-Myc, a proto-oncogene involved in cell cycle progression, apoptosis, and cellular transformation. Low c-Myc inhibits uncontrolled DNA synthesis associated with cancer and thus lowers the expression of PGM, PKM, and LDHA, and eventually downregulates carbon consumption needed to sustain malignancy. Lactucin also binds to PDH, reducing its concentration, and decreasing Pyruvate to Acetyl CoA conversion, thus downregulating the TCA cycle and lactate production. Thus, Lactucin inhibits cell proliferation and simultaneously increases cell cycle arrest in lung adenocarcinoma by reducing the MAPK/ERK and lowering carbon metabolism-related enzyme availability.
