AF selectively kills cancer cells with minimum toxicity to normal cells
We first tested the sensitivity of various cancer cell lines of different tissue origins to AF. This drug exhibited potent growth inhibition on all 33 cancer cell lines tested, with IC50 values of less than 2 µM in most cases (Fig. 1a). AF was particularly effective against lymphoma cells, including mantle cell lymphoma (MCL) JeKo-1 cell line, primary lymphoma cells isolated from patients with MCL and diffused large B cell lymphoma (DLBCL) (Fig. 1b). Notably, AF at low concentrations (0.2–0.5 µM) was highly effective against the malignant cells. In contrast, AF induced minimal cytotoxicity in non-cancerous cells, including primary lymphocytes isolated from the biopsy samples of patients with benign lymphadenitis (Fig. 1b), normal peripheral blood mononuclear cells (PBMC) and normal stromal cells (Fig. 1c and Supplemental Fig. 1a-b).
AF is highly effective against lymphoma cells in the presence of stromal cells
Since previous studies showed that stromal cells in tumor microenvironment could protect leukemic cells and promote drug resistance (10–13), we tested the effect of AF against lymphoma cells in the presence of stromal cells using a co-culture system. Surprisingly, instead of seeing a protective effect, we found that AF exhibited greater cytotoxic activity against lymphoma cells (both JeKo-1 and Raji) when they were co-cultured with NKtert stromal cells (Fig. 2a-b). These data together show a striking ability of AF to kill malignant cells in stromal microenvironment.
Inhibition of TrxRs by AF is not a key mechanism responsible for cytotoxicity
It has been suggested that inhibition of TrxRs by AF might lead to ROS-mediated apoptosis (14). We tested the impact of AF on TrxR activity and cell viability in two lymphoma cell lines, and found that AF could indeed inhibit TrxR activity and induced an increase of ROS (Fig. 3a). Pretreatment of cells with an antioxidant N-acetyl-L-cysteine (NAC) seemed able to reduce AF-induced apoptosis (Fig. 3b and Supplementary Fig. 2a), suggesting a potential role of ROS in drug-induced cell death. However, since NAC is a thiol-containing compound capable of directly conjugating with certain compounds including AF (15, 16), we tested if the protective effect of NAC was due to its conjugation with AF to prevent cellular uptake of AF. Quantitative assay of cellular gold (Au) content showed an 80% decrease of intracellular AF in the presence of NAC (Fig. 3b and Supplementary Fig. 2b). When the pre-incubated NAC was removed by washing before addition of AF to avoid direct NAC-AF binding in the medium, such washing restored cellular uptake of AF (Fig. 3b and Supplementary Fig. 2b) and almost completely abolished the NAC protective effect against AF cytotoxicity (Fig. 3b and Supplementary Fig. 2a). Since NAC pre-incubation followed by washing still led to lower cellular ROS (data not shown), its failure to prevent AF-induced cytotoxicity suggested that ROS accumulation might not be a key cytotoxic mechanism.
To further evaluate the role of TrxR inhibition in AF-induced cytotoxicity, we tested if suppression of TrxR expression by siRNA could affect cell viability. A partial silencing of TrxR1 expression by siRNA in Raji cells resulted in approximately 50% decrease of total TrxR enzyme activity, which was comparable to the degree of TrxR inhibition by AF, did not cause any loss of cell viability (Fig. 3c). Further, siRNA silencing of both TrxR1 and TrxR2 in a more readily transfectable cell line (HCT116) with a 75% reduction of total TrxR activity still did not induce any loss of cell viability (Fig. 3c). These data together suggest that inhibition of TrxRs was unlike a key event responsible for the cytotoxic effect of AF.
AF induces severe ATP depletion through double impacts on energy metabolism
To explore the potential mechanisms for AF-induced cytotoxicity in lymphoma cells, we first tested potential impact of AF on cellular energy metabolism, and observed that AF at a relatively low concentration (1 µM) could induce a rapid depletion of ATP in JeKo-1 and Raji cells (Fig. 4a and Supplementary Fig. 3). We then examined the effect of NAC on AF-induced ATP depletion. A simultaneous incubation of NAC with AF could prevent ATP depletion, whereas pretreatment of cells with NAC followed by washing before adding AF was unable to prevent ATP deletion (Fig. 4b). These results were consistent with the cytotoxicity data shown in Fig. 3b and Supplementary Fig. 2a and suggested that ROS did not play a key role in AF-induced energy collapse.
To further explore the mechanism for AF-induced ATP depletion, we tested the impact of AF on two main ATP generation pathways, oxidative phosphorylation and glycolysis, using a Seahorse extracellular flux analyzer to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). A substantial decrease of OCR was detected within minutes after addition of AF to JeKo-1 cells (Fig. 4c). This was consistently observed in other lymphoma cells (Supplementary Fig. 4). Concomitantly, a 30% decrease of ECAR was also observed after AF treatment in lymphoma cell lines (Fig. 4d and Supplementary Fig. 4). Silencing of TrxR expression by siRNA did not affect the effect of AF on oxygen consumption, since the control Raji cells and TrxR-silenced cells exhibited similar degrees of OCR inhibition by AF (Fig. 4e).
We then further examined the impact of AF on mitochondrial respiratory chain activity. Functional analysis revealed that AF caused a significant suppression of respiratory complex II (Fig. 4f) and a moderate decrease in complex III activity (Fig. 4g), whereas no changes in complexes I, IV and V were detected (Supplementary Fig. 5a). Western blot analysis showed no significant changes in expression of any respiratory chain proteins (Supplementary Fig. 5b). We then used specific inhibitors of mitochondrial respiratory complexes in combination with relevant respiratory substrates to analyze the detail changes in each mitochondrial respiratory chain activity. Supplement of the complex II substrate (succinate) resulted in an increase of OCR in the control cells but not in the AF-treated cells (Fig. 4h), indicating that the activity of complex II was inhibited by AF. In contrast, complex I inhibitor (rotenone) and complex III inhibitor (antimycin) caused a decrease of OCR in both control and AF-treated cells (Fig. 4h and Supplementary Fig. 5c), suggesting complexes I and III were not significantly affected by AF. Since the total mitochondrial oxygen consumption represents the electron transport activities from complex I→CoQ→III→IV and complex II→CoQ→III→IV (17), these data together suggest that the main site inhibited by AF was complex II.
To test if AF inhibition of mitochondrial respiration was responsible for its cytotoxicity, various concentrations of rotenone (a potent mitochondrial respiration inhibitor) were titrated to select a concentration that caused a similar degree of respiratory suppression induced by 1 µM AF (Supplementary Fig. 6a). This concentration of rotenone (0.05 µM), although potently inhibit respiration, did not cause any significant ATP depletion or cell death (Supplementary Fig. 6b and c). Importantly, the rotenone-treated cells exhibited a significant upregulation of glycolysis whereas the AF-treated cells exhibited a decrease in glycolysis (Fig. 4i), indicating that cells were able to compensate the energy loss from mitochondrial inhibition by rotenone, but not able to do so when they were treated with AF. These data together suggest a possibility that AF might inhibit both mitochondrial respiration and glycolysis leading to severe ATP depletion.
We then tested the impact of AF on several key glycolytic enzymes, including hexokinase (HK), glyceraldehyde phosphate dehydrogenase (GAPDH), pyruvate kinase (PK), and lactate dedydrogenase (LDH). A dose-dependent decrease in GAPDH activity was observed in two lymphoma cell lines treated with AF (Fig. 4j and Supplementary Fig. 7a). In vitro enzyme assay using purified GAPDH showed that AF directly inhibited GAPDH (Fig. 4k), whereas it had no impact on other glycolytic enzymes (Supplementary Fig. 7b-d).
Since pyruvate is the product of glycolysis that functions as a precursor for acetyl CoA production in mitochondria (18, 19), it might be possible that the suppression of mitochondrial respiration in AF-treated cells could be a consequence of glycolytic inhibition leading to a depletion of pyruvate. To exam this possibility, we tested whether exogenous pyruvate could rescue the AF-induced respiratory inhibition and cytotoxicity. Addition of pyruvate did not prevent AF-induced mitochondrial dysfunction (Supplementary Fig. 8a), nor did it suppress apoptosis (Supplementary Fig. 8b), indicating that AF-induced mitochondrial dysfunction was not a consequence of GAPDH inhibition.
Normal cells survive AF inhibition of mitochondrial respiration through upregulation of glycolysis
We then tested the impact of AF on energy metabolism in normal cells. Interestingly, mitochondrial respiration in normal PBMCs and stromal cells was inhibited by AF (Fig. 5a) to similar degrees as observed in cancer cells (Fig. 4c). However, unlike cancer cells whose glycolysis was also inhibited by AF, normal cells exhibited a significant upregulation of glycolysis after AF treatment (Fig. 5b). Such increase in glycolysis seemed able to compensate the decrease of mitochondrial ATP generation, since total cellular ATP did not decrease in the three types of normal cells treated with AF (Fig. 5c). The ability of normal cells to maintain cellular ATP seemed consistent with their ability to survive AF treatment.
Stromal cells promote TrxR1 expression in cancer cells and enhance AF-induced ROS generation
The finding that AF induced severe ATP depletion could explain its cytotoxic effect against cancer cells, but this could not explain why AF was more effective in killing cancer cells in the presence of stromal cells. In our attempt to explore the underlying mechanisms, we observed that in the presence of stromal cells, AF induced higher ROS generation compared to the cells treated with AF without stromal cells (Supplementary Fig. 9). Such higher ROS was unexpected, since stromal cells normally protect cancer cells by reducing their ROS stress (20). This unexpected observation prompted us to investigate the underlying mechanism. Since AF could inhibit TrxRs and causes ROS increase, we first examined the impact of stromal cells on TrxR expression in cancer cells. Western blot analysis revealed that TrxR1 expression was consistently up-regulated in lymphoma cells when they were co-cultured with stromal cells (Fig. 6a).
The consistent upregulation of TrxR1 by stromal cells led us to speculate its potential role in AF-induced ROS generation. Since the reactive cysteine and selenocysteine residues in the active site of TrxR1 play key roles in transporting electron and mediating the redox cycle during the reduction of thioredoxin (21), we postulate that a reaction of AF with the cysteine or selenocysteine in the active site of TrxR1 would disrupt the electron transport process, leading to a leak of electrons to be captured by molecular oxygen to form superoxide as illustrated in Fig. 6b. As such, AF would turn TrxR1 into a ROS-generating protein, and the higher expression of TrxR1 in cancer cells in stromal environment would generate more ROS in the presence of AF. To test this possibility, we used a luminescence-based in vitro assay to measure superoxide generation in a cell-free system containing purified TrxR1, Trx, NADPH, and AF. Addition of AF to the complete reaction mixture caused a rapid generation of ROS in a time-dependent manner (red color curve). Removal of AF or TrxR1 from the reaction system led to a substantial decrease in ROS generation rate (Fig. 6b), suggesting that both TrxR1 and AF were required for a sustained production of ROS. Collectively, these data suggest that AF could turn the TrxR1/Trx/NADPH redox system into a ROS-generating machinery, and the increased expression of TrxR1 in cancer cells in stromal environment renders them more prone to AF-induced ROS generation.
AF exhibits potent therapeutic activity in lymphoma models
Since AF could effectively kill lymphoma cells in vitro, we further test its therapeutic activity in vivo. In the lymphoma xenograft mouse model bearing lymphoma cells, AF treatment showed a significant therapeutic effect (Fig. 7a). This drug was well tolerated, and the mice exhibited no significant loss of body weight (Fig. 7a). Importantly, there was a significant decrease in both OCR and ECAR in tumor cells isolated from the lymphoma-bearing mice treated with AF (Supplementary Fig. 10), indicating that AF was able to inhibit tumor cell energy metabolism in vivo, similar to that observed in vitro. These data together suggest that AF has promising in vivo therapeutic activity in lymphoma.