Curative effect of RGE ip injection against EL4-induced ascites

EL4 is a line of highly aggressive mouse lymphoma cells that induces rapid and lethal ascites in mice as described previously31. In this experiment, all the syngeneic C57BL/6 mice were given intraperitoneal (ip) injection with 2 × 106 EL4 cells from the cell culture. Twenty-four hours later, the injected mice were divided into the groups of RGE ip injection as treatment and the control without RGE injection. For the treated group, each mouse of average 15 g body weight was given an ip injection of 1 ml RGE that was equivalent to 100 mg wet weight of raw garlic. The control group was left untreated. The ip injection of RGE was repeated daily for 21 days. As shown in Fig. 1, all eight mice in the control group, as expected, developed ascites that increased the body weight significantly higher than the mice without ascites (Fig. 1a). The ascites development was markedly visible (Fig. 1b, c). All the mice in the control group were moribund as indicated in the figure and were killed. However, the mice given the ip injection of RGE were healthy without any sign of adverse effect (Fig. 1b, c). Moreover, when the injection of RGE stopped between days 22 and 37 (or longer up to four more weeks, data not shown), the mice remained healthy, indicating there was no re-growth of ascites even long after the RGE treatment stopped. To assure that the failure to develop ascites in these mice was solely due to the garlic treatment and not due to other unknown factors, on day 38 all the mice of the previously RGE treated group were again given the ip injection of 2 × 106 EL4 cells and left without the injection of RGE. Re-challenge with EL4 cells developed ascites in all the mice (Fig. 1a), indicating that the failure of ascites development was entirely due to the RGE treatment.

Fig. 1: The therapeutic activity of RGE injection against EL4-induced ascites. Ten C57BL6 mice were inoculated ip with 2 × 106 EL4 (mouse ascites lymphoma cells). One day later, five of these mice were treated daily by the ip injection of 1 ml RGE (equivalent to 100 mg wet weight of raw garlic) between day 1 and day 22. The treatment was stopped for all the five mice between day 23 and day 37. The mice were re-injected with 2 × 106 EL4 ip and left untreated until moribund. a The body weight gain in grams for reflection of ascites development. b Survival curves of the mice in different groups. c Representative images of the mice with and without ascites Full size image

Curative effect of RGE ip injection, not ingestion, against S180-induced ascites

S180 is another line of mouse aggressive lethal sarcoma cell. The survival time (average 15 days) was slightly longer than that of EL4 (average 13 days) when the same doses were given ip. All the mice were given ip injection with a dose of 2 × 106 S180 cells. One day later, the mice were divided into five different groups. Three groups were treated daily with three different preparations of RGE: RGE, the aqueous phase and the organic phase of RGE, at a dose equivalent to 100 mg of the original raw garlic wet weight. One group was given RGE orally via gavages with the daily doses equivalent to 100 mg wet weight of garlic for each mouse. One group was left untreated as the control. As expected, all the mice in the control group developed ascites and were euthanized after moribund (Fig. 2a, b, c). Similarly, all the mice in the group treated with RGE given via gavages developed ascites without any increase in the survival time. Moreover, all the mice in the group treated with the aqueous phase of RGE given via ip also developed ascites without any significant increase in the survival time. In a sharp contrast, when RGE or the organic phase of RGE was given ip at a same daily dose for 21 days, all the mice in these two groups remained healthy without any sign of adverse effect.

Fig. 2: The therapeutic activity of RGE injection against S180-induced ascites. Thirty-one C57BL6 mice were inoculated ip with 2 × 106 S180 cells (mouse sarcoma cells). One day later, five mice were given 1 ml RGE via daily oral gavage between day 1 and day 22. Eight mice were treated by the ip injection of 1 ml RGE (equivalent to 100 mg wet weight of raw garlic) daily between day 1 and day 22. Five mice were treated by the ip injection of 1 ml aqueous phase of RGE (equivalent to 100 mg wet weight of raw garlic) daily between day 1 and day 22. Five mice were treated by the ip injection of 1 ml organic phase of RGE (equivalent to 100 mg wet weight of raw garlic) daily between day 1 and day 22. a The body weight gain in grams for reflection of ascites development. b Survival curves of the mice in different groups. c Representative images of the mice with and without ascites Full size image

Comparison of RGE with the extracts from other fruits and vegetables for their anticancer activities

To compare the anticancer property of RGE, we prepared extracts of additional seven vegetables and 14 fruits in the way similar to that of RGE preparation. All the dilution factors were normalized to their respective original wet weights. All the extracts were added individually to the cell cultures of three different human cancer cell lines for direct comparison: Hela (human cervical carcinoma), 5637 (human bladder carcinoma), and J82 (human bladder adenocarcinoma). The cancer cells were plated at 15% confluence and grew for 24 h at 37 °C before the juices were added to a 1/200 dilution factor of their respective wet weights (5 mg/1 ml). The pH values of the final solutions were unaffected as the volumes of the added extracts were relatively small and the culture medium color indicator of pH remained unchanged till the end of cell culture. The cells were co-cultured for another 24 h at 37 °C before the viable cells in each well were analyzed with CCK-8 kit. As seen in Fig. 3, RGE was the only one that killed all three lines of cancer cells completely. We also tested RGE against additional cancer cells and found that it was equally effective in killing all other cancer cells tested so far, including but not limited to EL4 and S180 (data not shown). The anticancer property of RGE was in agreement to the previous study on eight cancer cell lines (PC-3, AGS, U-87, DAOY, MCF-7, A-549, Panc-1, and Caki-2) at a much higher concentration (166 mg/ml)26. RGE was also shown to be highly effective in killing above eight cancer cell lines 100%. In addition, the extracts from cauliflower, red grape, guava, and strawberry exhibited equal effectiveness against Hela cells but much less-killing activity against 5637 and J82 (Fig. 3). RGE had stronger anticancer property against various types of cancer cells in cell cultures in comparison with the extracts from other vegetables and fruits tested so far.

Fig. 3: In vitro killing of human cancer cells by co-culturing with the extracts of different fruits and vegetables. All extracts of fruits and vegetables were prepared in the same manner and similarly diluted to 1/200 of their respective wet weights (5 mg/ml) for co-culturing with Hela (human cervical carcinoma) and human bladder cancer cells 5637 and J82. After 24 h in the cell culture condition at 37 °C, the percentages of survived cancer cells were measured by CCK-8 Full size image

Size fractionation of the RGE anticancer activity

To roughly estimate the size(s) of the active molecules involving the anticancer property, we partitioned RGE through a molecular size filter, via which the molecules smaller than 3000 Dalton went through the membrane and the molecules larger than 3000 Dalton were retained at the top chamber of the filter. After centrifugation, most yellowish color and viscosity remained in the top fraction, whereas the lower fraction was clear and not viscous. The fractions were brought back to the original volumes by distilled water. The size fractions and RGE were added to the cell cultures of five different cancer cell lines and co-cultured for 24 h at 37 °C. Viable cells were analyzed by CCK-8 kit. The results in Fig. 4 show that the anticancer property of the ≤ 3K fraction was nearly identical to that of RGE at all the dilutions against all five cancer cell lines. On the other hand, the anticancer property was completely absent from the ≥ 3 fraction against the same cell lines. In addition, the 1/200 dilution (5 mg/ml) seemed to be a critical concentration, via which the least amount of garlic achieved the maximum effect.

Fig. 4: Distribution of cancer cell killing activity in the size fractions of RGE. RGE was fractionated with a size filter membrane of 3000 Dalton cutoff into the fraction smaller than 3000 Dalton (< 3 K) and the fraction larger than 3000 Dalton (> 3 K). The fractions were co-cultured with five different kinds of cancer cells at 37 °C for 24 h at the indicated dilutions. Survived cells were measured by CCK-8 Full size image

Solubility fractionation of the anticancer activity in RGE

To determine the solubility of the anticancer property, we partitioned RGE into the organic phase and aqueous phase via chloroform extraction. The aqueous phase was brought back to the original volume by phosphate-buffered saline (PBS) and the organic phase was reconstituted back to the original volume by 5% dimethyl sulfoxide (DMSO). At the indicated dilutions, the different RGE preparations were co-cultured individually with five different cancer cell lines for 24 h. As shown in Fig. 5, all anticancer properties of RGE were present in the organic phase. Instead of having the anticancer property, the aqueous phase of RGE even stimulated the growth of S180 and especially K562 human leukemia cells to onefold more than RGE.

Fig. 5: In vitro killing of five different cancer cell lines by aqueous phase and organic phase of RGE. The aqueous phase and organic phase of RGE were prepared as described in the section of methods. After co-culturing with the indicated fractions of RGE at 37 °C for 24 h, survived cells were measured by CCK-8 Full size image

RGE induced apoptosis in cancer cells

To examine the mechanisms of the induced cancer cell death, S180 cells after being treated by various RGE preparations were examined morphologically. After treated by RGE or the organic phase, all S180 cells showed the highly condensed cellular and nucleus morphology, being consistent with apoptosis (Fig. 6). However, S180 cells treated with the aqueous phase or 5% DMSO did not show any sign of cell death.

Fig. 6: Morphologies of the S180 cells treated by RGE and its different fractions of hydrophobicity. The aqueous phase and organic phase of RGE were prepared as described in the methods section. After co-culturing with the indicated fractions of RGE at 37 °C for 24 h, cells were spun down onto glass slides, stained with Diff-Quik and examined and photographed with light microscope Full size image

Partial inactivation of RGE anticancer activity by heat

As a flavoring food, garlic is often ingested after cooking. Many studies of the anticancer properties were also done with the cooked garlic. To determine its sensitivity to heat, we heated RGE at 100 °C for 10 min. As shown in Fig. 7, the heat-treated RGE was compared with the unheated RGE for their anticancer properties against five different cancer cell lines. At 1/100 dilution, the heat treatment did not alter the anticancer activity of RGE against J82 and Hela, but reduced the anticancer properties partially against 5637, S180, and K562. At 1/200 dilution, the cancer killing activities against J82, 5637, Hela, and K562 were mostly, and against S180 was partially, abolished by the heat treatment. These results indicate that some anticancer activities of RGE were heat-labile and others were heat-resistant.

Fig. 7: Heat sensitivity of RGE anticancer activity against five different cancer cell lines. The heated RGE was heated in a metal heating block at 100 °C for 10 min. After co-culturing with RGE or the heated RGE at 37 °C for 24 h, the survived cells were measured by CCK-8 Full size image

High selective killing of RGE against cancer cells but not normal cells

Our results showed that the injected RGE had no adverse effect on the treated animals, indicating that RGE did not harm normal cells. We wanted to further verify this ability of RGE to distinguish cancer cells from normal cells in the in vitro system. We isolated human polymorphic nucleus (PMN) cells from the blood of healthy donors. Human PMN and human cervical carcinoma Hela cells were co-cultured with RGE at different dilutions. At 1:400 or higher dilutions of RGE, there was no specific cytotoxicity against PMN or Hela (Fig. 8). At 1:200 or lower dilutions nearly all Hela cells were killed and most PMN were alive. The apparent selectivity against cells in vitro was in an agreement to that of the in vivo results of the injected RGE.