In Vitro Effect of Herbal Medicines with Thermal Characteristics on Heat Sensitivity in Cancer Cells
Article information
Abstract
Background
Cancer remains a major public health threat even though there have been breakthroughs in conventional diagnostics and therapies. Alternatively, treatment with mild hyperthermia and herbal medicine treatment [selected using traditional Korean medicine theory 4 qi (cold, cool, warm and hot) and using the 5 senses of taste (sour, bitter, sweet, pungent and salty)], may be an option to promote cancer cell death in patients where the cancer is accessible.
Methods
To investigate effect of combination treatment of herbal medicines and hyperthermia in vitro on cancer cell lines (ACHN, AGS, A549 and U937), the qualities of 38 medicinal herbs and sensitivity to mild hyperthermia (42 and 43°C) treatment were examined. An assay was performed to detect cell viability and proliferation (MTT) following exposure to medicinal herbs and hyperthermia.
Results
Heat sensitizing herbal medicines were determined to be larger in the warm and hot groups of medicinal herbs (29.6%) than the groups which were neither warm nor hot (18.2%). In addition, the proportion of heat sensitizing effect with bitter and pungent flavors was 33.3 % and 32.1 %, respectively, greater than the average (26.3 %).
Conclusion
In conditions of mild hyperthermia in cancer cell lines, incubation with herbal medicines caused cancer cell death in vitro. These results suggest that the use of traditional herbal medicines with warm, hot, pungent and bitter characteristics may be a useful treatment for cancer using conditions to induce mild hyperthermia and this requires further investigation.
Introduction
The second leading cause of mortality in the United States (US), behind ischemic heart disease, is cancer [1,2]. Despite significant progress in screening, prevention, and treatment, the incidence of cancer continues to rise. The prevalence of cancer is anticipated to rise progressively as the average age of the general population increases. In addition, late diagnosis contributes to a higher death rate in low-income nations. The high cost of cancer therapy also restricts access to cancer remedies.
Although several cancer therapies exist and have been extensively explored, conventional cancer therapy techniques have major shortcomings, including recurrence of the cancer, side effects, and high cost [3]. For instance, cachexia, a side effect of several chemotherapies, is related to mortality. To compensate for the limitations of conventional cancer therapy, hyperthermia has started to emerge as a viable method for cancer treatment [4,5]. Hyperthermia indicates either an abnormally high fever or refers to the induction of fever to treat a disease. Hyperthermia is a potential option for the treatment of cancer since it has less side effects than conventional medications and is cost-effective in light of its substantial therapeutic benefit, particularly when paired with other treatments [6,7]. It has been reported that hyperthermia alone may not be adequate to eradicate cancerous cells but in combination with natural products, the limitations of hyperthermia may be overcome [8]. Thermic stimulation causes apoptosis but also activates transcription factors important in tumor survival, causing tumor cells to proliferate more rapidly and develop resistance to anticancer drugs. To complement these apoptotic qualities, hyperthermia is often combined with other therapeutic procedures like radiation, chemotherapy, surgery, and immunotherapy [9]. Specifically, natural products may significantly improve the outcome of hyperthermia on cancer cells by acting synergistically and with fewer adverse effects, and in addition is inexpensive [10].
In traditional Korean medicine, there is a theory called 4 qi (cold, cool, warm, and hot) and 5 tastes (sour, bitter, sweet, umami and salty). The hypothesis is that herbal medicine contains the properties of both hot and cold and five tastes. Here, we proposed that the warm properties of medicinal herbs help increase the effectiveness of thermal therapy. We studied whether herbal medicines with warm properties increase the sensitivity to thermal therapy. Through the aforementioned research, we aim to identify a variety of medicinal herbs that may mitigate the disadvantages of mild hyperthermia treatment. Experiments will also reveal how the 4 qi and 5 tastes theory suggested by traditional medicine operates in in vitro experiments.
Materials and Methods
1. Reagents
Medicinal herbs (Kwangmyeongdang Medicinal herbs, Ulsan, Republic of Korea) were thoroughly ground and homogenized. The extract was soaked for 24 hours at room temperature in 70% ethanol. To obtain a sample, the resulting extract was lyophilized, concentrated under decreased pressure, and filtered (pore size: 5 μm). Dimethyl sulfoxide (DMSO; Samcheon Chemical, Seoul, Korea) solutions of 100 and 200 μg/mL were prepared and stored at 4°C. The primary selection criteria for a herbal medicine was that it was a drug that was clinically well-known, and had similarities in properties to other effective drugs. In the non-treatment group, the same amount of DMSO was applied to the cancer cells as in the treatment group.
2. Cell culture
Cancer cell lines (ACHN, AGS, A549 and U937 cells; Korean Cell Line Bank, Seoul, Korea) were maintained in medium (RPMI or DMEM) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and 1% penicillin-strep (Gibco, Grand Island, NY, USA) and incubated at 37°C in a humidified incubator containing 5% CO2.
3. Hyperthermia
For this experimental model of mild hyperthermia the temperatures 42°C and 43°C were selected [8]. Cancer cells were seeded in a 96-well plate at a density of 1.5 × 104 cells/well in 0.1 mL of media, followed by immersion in a temperature-controlled water bath (42°C or 43°C for mild hyperthermia and 37°C for normothermia) for 30 minutes. Medicinal herbs were used at the indicated concentrations was added to the samples of cancer cells 1 hour prior to hyperthermia treatment.
4. MTT assay
The MTT assay was performed to detect cell viability and proliferation following exposure to medicinal herbs and hyperthermia. Cancer cells were seeded in 96-well plates (100 μL/well) at a density of 1.5 × 104 cells/mL and allowed to adhere overnight. Then, various concentrations of medicinal herbs were added and the plates which were incubated at 37°C for 1 hour in a humidified atmosphere containing 5% CO2, followed by immersion in a temperature-controlled water bath at 37°C or 43°C for 30 minutes. After 48 hours of incubation at 37°C, 5% CO2, 20 μL of MTT (2 mg/mL in PBS; AMRESCO, Solon, OH, USA) was then added to each well and cultured for another 2 hours. Following this, the culture media were removed, and the cells were lysed in 100 μL of DMSO. Absorbance was measured with an automated spectrophotometric plate reader at a wavelength of 570 nm. Cell viability was normalized with untreated controls.
5. Statistical analysis
All numeric values are represented as the mean ± SD. Statistical significance of the data compared with the untreated control was determined using the unpaired Student t test. p values of p < 0.05, p < 0.01 and p < 0.001 were determined as statistically significant.
Results
1. H1, H2 and H3 sensitized cancer cells to hyperthermia
Four medicinal herbs with hot characteristics were selected and examined to determine whether the herbs could boost cancer cell’s sensitivity to cancer hyperthermia treatment. Three of the four herbal medicines sensitized cancer cells to hyperthermia. The names of the medicinal herbs were numbered according to their properties, however the names of the herbs that were proven to be effective in this study were withheld since additional research is now being conducted. H1 sensitized Human Renal adenocarcinoma cell line (ACHN) cells to hyperthermia of 43°C therefore H1 and hyperthermia synergistically induced cell death in ACHN cells (Fig. 1). H2 also increased sensitivity of ACHN to hyperthermia of 42 and 43°C (Fig. 1). H3 has shown its heat sensitizing effect in a pro-monocytic, human myeloid leukaemia cell line (U937; Fig. 1).
Heat-sensitizing effect on cancer cell lines induced by hyperthermia and of medicinal herbs capable of inducing hot characteristics.
* p < 0.05.
** p < 0.01.
*** p < 0.001 vs. 37 °C control group; ## p < 0.01, ### p < 0.001 vs. hyperthermia treatment group.
H, hot medicinal herb.
Cancer cell lines (ACHN, AGS, A549 and U937 cells) were treated with hot medicinal herbs (H1–3) and hyperthermia of 42°C and 43°C, then incubated for 24 hours. Representative results of H1 (ACHN), H2 (ACHN) and H3 (U937) are shown. Cell viability was measured by the MTT assay. Herb names were anonymized and marked as H1, H2, and H3.
2. W1-5 sensitized cancer cells to hyperthermia
There were 23 medicinal herbs with warm properties selected and tested to determine whether cancer cell sensitivity to hyperthermia treatment could be increased. Five of the 23 selected medicinal herbs enhanced susceptibility to hyperthermia in the cancer cells. The names of the medicinal herbs were numbered according to their properties, however the names of the herbs that were proven to be effective in this study were withheld since additional research is now being conducted. W1 sensitized non-small-cell lung cancer cell line (A549) cells to hyperthermia using 42 and 43°C (Fig. 2A). W2 also increased sensitivity of ACHN to hyperthermia of 42 and 43°C (Fig. 2B). W3 dramatically increased heat sensitivity of U937 cells and synergistically induced cell death (Fig. 2C). A combination of hyperthermia with W4 or W5 (Figs. 2D and 2E) inhibited cell viability in a synergistic manner.
Heat-sensitizing effect on cancer cell lines induced by hyperthermia and medicinal herbs capable of inducing warm characteristics.
* p < 0.05.
** p < 0.01.
*** p < 0.001 vs. 37°C control group; ## p < 0.01, ### p < 0.001 vs. hyperthermia treatment group.
W, warm medicinal herbs.
Cancer cell lines (ACHN, AGS, A549 and U937 cells) were treated with warm medicinal herbs (W1–W5) and hyperthermia at 42°C and 43°C, then incubated for 24 hours. Representative results of W1 (A549), W2 (ACHN), W3 (U937), W4 (AGS) and W5 (AGS) are shown. Cell viability was measured by the MTT assay. Herb names were anonymized and marked as W1, W2, W3, W4 and H5.
3. C1 and N/A1 sensitized cancer cells to hyperthermia
We evaluated seven medicinal herbs that were neither hot nor warm for their capacity to boost cancer cell sensitivity to hyperthermia. Two of the seven medicines increased the vulnerability of cancer cells to hyperthermia. The names of the medicinal herbs were numbered according to their properties; however, the names of the herbs proven to be effective in this study were withheld due to ongoing research. C1 that has cold properties increased the effect of hyperthermia on human gastric cancer cell line (AGS) cells (Fig. 3A). N/A1 medicinal herb is well known for having a little sweet flavor and being neither cold nor hot. N/A1 also sensitized AGS cells to hyperthermia of 43 °C (Fig. 3B).
Heat-sensitizing effect on cancer cell lines induced by hyperthermia and medicinal herbs either capable of inducing cold characteristics (C1), or neutral characteristics [neither cold nor hot (N/A1)].
* p < 0.05.
** p < 0.01.
*** p < 0.001 vs. 37°C control group; ## p < 0.01, ### p < 0.001 vs. hyperthermia treatment group.
C, cold medicinal herbs; N/A, neither cold nor hot medicinal herbs.
Cancer cell lines (ACHN, AGS, A549 and U937 cells) were treated with medicinal herbs (C1 and N/A1) and hyperthermia at 42°C and 43°C and incubated for 24 hours. Representative results of C1 (AGS) and N/A1 (AGS) are shown. Cell viability was measured by the MTT assay. Herb names were anonymized and marked as C1 and N/A1.
4. Herbal medicines with warm properties were more likely to promote heat sensitivity
We studied the effect of 38 herbal medications on heat sensitivity and determined that 10 showed a heat sensitizing effect. Next, we investigated the 4 qi and five tastes of the 10 successful herbal medications (Table 1). Along with the drug’s name, the attributes of the 28 ineffective herbal medications are listed in Table 2. the proportions of effective medications based on 4 qi and five flavors are summarized in Table 3. The proportion of herbal medications possessing heat-sensitizing effects with warm or hot qualities was substantially distinct from the proportion of medicines without warm properties. Although the proportion of medications with hot or sour flavor was high, we believe that a sufficient number of substances should be investigated further. Interestingly, it turns out that the bitter and pungent tasting herbal medicines were more potent.
Qi and Flavor Characteristics of 10 Heat-sensitizing Medicinal Herbs.
Qi and Flavor Characteristics of 28 Herbs without Heat-sensitizing Effects.
Percentage of Heat-sensitizing Herbs Based on Their Qi and Flavor Characteristics.
Discussion
Cancer accounts for around 1.6 million deaths annually [5,11]. Although recent progress has been encouraging, many obstacles remain in the treatment of cancers. Hyperthermia is one potential method for promoting cancer cell death. Physiological reactions of the body to high-temperature stimulation include alterations in membrane permeability or the cytoskeletal system, macromolecule synthesis or intracellular signaling, and DNA repair inhibition [12,13]. In addition, in vitro studies on the combination of hyperthermia with natural products have demonstrated the efficacy of using compounds produced from nature in conjunction with hyperthermia [10,14]. Therefore, we hypothesized that the combination of herbal medications capable of inducing warm or hot qi and hyperthermia might boost the efficacy of hyperthermia treatment of cancer in vitro. There were 38 herbal medicines with or without warm or hot qi qualities chosen to determine if they could boost the sensitivity of hyperthermia therapy.
There were 4 hot medicinal herbs and 23 warm medicinal herbs. The extract was obtained in 70% ethanol since it is a more effective solvent than alcohol for extracting medicinal compounds from herbs because it is a more polar solvent. So, there were 27 drugs with warm properties and 11 drugs without warm properties. Initially, we attempted to determine the relationship between warm herbal medicine and thermal therapy, so a larger number of warm herbal medicinal drugs were used. Also, among medicinal herbs, hot medicines are much less prevalent than warm medicines, thus we were not able to test many hot medicines. The proportion of heat sensitizing medicinal herbs in the warm and hot groups was 29.6% which was larger than where the groups were neither warm nor hot groups (18.2%). In conclusion, we have determined that the warming properties of traditional herbal medicine can have a thermal effect on cancer line cells.
There were four herbal medications among the experimental drugs capable of inducing warm qualities, and three of them increased thermal sensitivity. Since there are few drugs in herbal medicine capable of inducing hot qualities, only four drugs have been evaluated. Therefore, we believe further research is required on herbal medicines with hot properties. In addition to investigating the effects of 4 qi, the five different taste senses of medicinal herbs were also analyzed. The proportion of effective herbal medications with bitter or pungent flavors was 33.3% and 32.1%, respectively, greater than the average (26.3%). In addition, it was confirmed that 45.5% of medications having both bitter and pungent flavors increased the effect of sensitivity.
Conclusion
The increased life expectancy and quality of life in a human population leads to an increased number of elderly cancer patients and cancer-related deaths. Unlike other diseases, conventional medicines have not shown improved effects in the treatment of cancer. Thermal therapy has been reported to be an effective treatment for cancer that may compensate for the deficiencies of existing therapies. To this goal, it is vital to address the deficiencies of thermal cancer treatment. This study revealed that herbal medications capable of inducing warm qi, as established by traditional herbal medicine, can enhance the efficacy of thermal therapy. Through this in vitro study, it is feasible that studies in the future may lead to a new approach to cancer treatment which enhances the efficacy of hyperthermia treatment by employing the traditional medicine principles of warm qualities, bitter taste, and pungent flavor.
Notes
Author Contributions
Conceptualization: SHB, Formal analysis: CRA, Investigation: SJ and SK, Methodology: CRA and SHB, Validation: CRA, Writing – review and editing: SHB.
Conflicts of Interest
The authors declare no conflicts of interest.
Ethical Statement
This research did not involve any human or animal experiments.
Data Availability
All relevant data are included in this manuscript.
Funding
This work was supported by National Research Foundation of Korea grant funded by the Korea government (no.: NRF-2020R1I1A3063625).