Blog

Shikonin is a naturally occurring compound found in Gromwell roots, traditionally used in Chinese medicine for various purposes. Interestingly, shikonin and the related naphthoquinone compounds are responsible for the red/purple color of the roots. Studies have shown that shikonin itself has antibacterial, anti-inflammatory, and anti-tumor effects. Recently, a new study investigated the effects on brain tumors.

The difficulty in treating brain tumors is the presence of the blood-brain barrier and also the blood-tumor barrier. These barriers prevent most small molecules from crossing, and therefore brain tumors are protected from most chemical treatments. Furthermore, the current standard treatment for glioblastoma includes chemotherapy with temozolomide, however, the glioblastoma cells very quickly develop resistance to this chemical.

In vitro studies showed that shikonin does inhibit glioblastoma cells quite effectively. Therfore, an alternative method of delivery was attempted to cross the barriers. First, lab mice were infused with cancer cells directly into the thalamus. Next, direct intracranial infusion of shikonin was applied into the same spot. By this method, the blood-brain and blood-tumor barriers are physically passed through by the injection needle. In this way, the effect of the treatment in a living system can be observed.

For this study, 2 different brain cancer cell lines were used. The mice from either group, when treated with shikonin, showed significantly longer survival times. Some of the mice were treated with combination of shikonin and temozolomide or temozolomide alone. The effects of the combination treatments were inconclusive. Once cancer cell line seemed to have even better success with a combination treatment, but the other line did not. In addition, neither cancer line, when treated with temozolomide alone, showed significant improvement.

Although shikonin has poor bioavailability and cannot cross the blood-brain barrier on its own, most chemical treatments will face the same problem. Therefore, using direct intracranial infusion may help develop more effective treatment for brain tumors.

Maeoka, R, Ouji Y, Nakazawa T, et al. Local administration of shikonin improved the overall survival in orthotopic murine glioblastoma models with temozolomide resistance. Biomed Pharmacother. 2023 Oct:166:115296. PMID: 37557011

Oridonin is a naturally occurring chemical that can be extracted from the Chinese sage bush Isodon rubescens, formerly known as Rabdosia rubescens. It is used in traditional Chinese medicine to block cancer metastasis and recurrence and to overcome therapeutic resistance. Recently, a new study has been completed which looked at its effects on a lung cancer xenograft mouse model.

To begin, the mice were injected subcutaneously with human lung cancer cells. The tumors were allowed to grow and develop to a specified volume prior to beginning treatment. Once the tumors were developed, oridonin was injected intraperitoneally five times each week. In addition, NK-92MI cells were injected intravenously once a week. These “natural killer cells” are a naturally occurring part of an immune system. However, the mouse model used for this experiment does not have a functioning immune system. Therefore, the killer cells were injected during the study.

The results of the study showed that oridonin treatment enhanced the activity of the natural killer cells against all three different cancer cells lines studied. It was also revealed that ordidonin seems to be non-toxic to the liver and kidney. This is exciting because selectively harming only cancer cells without effecting normal cells always proves to be a challenge.

Understanding of the immunostimulatory effect of oridonin on NK cells more thoroughly may help to develop a new lung cancer treatment.

Hwang T, Chang C. Oridonin enhances cytotoxic activity of natural killer cells against lung cancer. Int Immunopharmacol. 2023 Sep;122:110669. doi: 10.1016/j.intimp.2023.110669. PMID: 37480753

Prostate cancer is the second most common cancer worldwide, leading to more than one million diagnoses per year. Metastatic prostate cancer is commonly treated with hormone therapy. These cancer cells need activation of the androgen receptor to divide, so drugs that drop testosterone levels block tumor growth. Unfortunately, resistance to hormone therapy tends to develop in one to two years.

Resistance often involves cancer cells reactivating the androgen receptor without needing testosterone. The authors of a recent study were interested in finding a way to re-inactivate it. The protein CDK7 is an attractive drug target because it is involved in both androgen receptor regulation and cell cycle regulation. The drug CT7001 inhibits CDK7, and shows promising experimental results in several other cancers. Therefore, these authors tested the effectiveness of CT7001 in prostate cancer.

They found that in prostate cancer cell lines, CT7001 binds to CDK7. This molecule also inhibits the androgen receptor, inhibits cell growth, and causes cell cycle arrest. The authors then treated mice carrying prostate cancer tumors with CT7001, or enzalutamide (a drug that blocks testosterone), or both. CT7001 shrank tumors, and the combination of CT7001 and enzalutamide worked even better.

The in vitro and mouse evidence from this study suggests that this chemical could help fight this disease, especially in combination with hormone therapy drugs.

Constantin T, Varela-Carver A, Greenland K, et al. The CDK7 inhibitor CT7001 (samuraciclib) targets proliferation pathways to inhibit advanced prostate cancer. Br J Cancer. 2023 Jun;128(12):2326-2337. PMID: 37076563. doi: 10.1038/s41416-023-02252-8

Okadaic Acid is an aquatic toxin that may be accumulated in the fatty tissues of shellfish and other fish. It is produced by microalgae, particularly troublesome during algal blooms, and also leads to contamination of the water itself. This compound is stable even when heated to high temperatures, so boiling or cooking contaminated water or food sources will not destroy the toxin. Consumption of these materials poses a risk to human health causing a variety of severe health problems.

Because there is no easy treatment for contaminated materials, many countries have set strict regulations for acceptable levels of this toxin in seafood. However, the traditional testing methods require highly complex labor-intensive and time-consuming sample preparation. In order to efficiently screen a large number of samples, a new method is necessary.

A New Testing Method

Therefore, a new polarization fluorescence immunoassay (FPIA) has been developed. This FPIA method uses a portable analyzer, requires only minimal sample preparation, and each test requires only 5-10 minutes to complete. First, a fluorescent tracer molecule selectively binds with okadaic acid. Then, this conjugate molecule interacts with specific anti-okadaic acid monoclonal antibodies. Several reference samples with known concentrations are analyzed, and then the actual sample is analyzed and compared.

The Traditional Testing Methods

Previous methods relied on either ELISA (enzyme-linked immunosorbent assay), requiring several hours and tedious sample preparation, or ICA (immunochromatographic analysis), requiring 10-40 minutes and more limited level of detection. In addition, these methods require the sample testing to be performed in a laboratory, which is less convenient.

Both climate change and pollution are leading to more frequent algal blooms in recent decades. Therefore, the ability to monitor for these toxins is becoming increasingly important.

Several of these reference compounds are available at LKT Labs including: Okadaic Acid, Okadaic Acid Ammonium, and Okadaic Acid Sodium.

Hendrickson O, Mukhametova L, Zvereva E, et al. A sensitive fluorescence polarization immunoassay for the rapid detection of okadaic acid in environmental waters. Biosensors (Basel). 2023 Apr 16;13(4):477. doi: 10.3390/bios13040477. PMID: 37185552

Cembrenediols are naturally occurring compounds found in fresh tobacco. However, the commercial tobacco products that many people know and love are purposely fermented to produce customized flavors, which degrades the cembrenediol. This is unfortunate, because while it is well-known that tobacco contains several carcinogens, cembrenediol is actually found to have anticancer activities.

A recent study on prostate cancer used a mouse model to demonstrate that treatment with cembrenediol prevented recurrence of this cancer. The mice were first implanted with prostate cancer cells, which were allowed to develop into tumors. The tumors were then removed, followed by 8 weeks of treatment with cembrenediol. At the end of the study the animals were sacrificed and the organs examined. The animals that had received cembrenediol treatment showed much lower chance of cancer recurrence compared to vehicle-treated animals.

Experiments were also run on human prostate cancer cell lines. The cells struggled to grow when treated with the cembrenediol. The treated cells showed lower migration capability and lower colony formation capability. Further evidence suggests that this treatment impacts the metabolism of these cancer cells.

Additionally, the cembrenediols and analogs have been found to be potent inhibitors for other cancers as well. Therefore, although tobacco is generally known for its cancer-causing potential, it may be possible that this plant, when processed and used another way, may provide effective cancer treatment.

Cembrenediols available at LKT Labs:

α-Cembrenediol

β-Cembrenediol

β-Cembrenediol Methyl Ether

Mudhish E, Siddique A, Ebrahim H, et al. The tobacco β-Cembrenediol: a prostate cancer recurrence suppressor lead and prospective scaffold via modulation of indoleamine 2,3-dioxygenase and tryptophan dioxygenase. Nutrients. 2022 Apr 4;14(7):1505. doi: 10.3390/nu14071505. PMID: 35406118

Vitamin K2 has several major forms due to slight differences in the side chain of the molecule. The major forms are menaquinone-4 (MK-4), menaquinone-7, menaquinone-8, and menaquinone-9. The major source of this vitamin is the conversion of vitamin K1 present in the diet. The presence of vitamin K directly impacts the synthesis and metabolism of sphingolipids in the brain. The sphingolipids in turn impact a variety of neuronal processes, including aging.

Therefore, the hypothesis was proposed, that vitamin K2 may provide some protection against age-related cognitive deterioration. This hypothesis was put to the test using Sprague-Dawley rats in an animal lab. Twenty rats were divided into two groups: (1) Control- sunflower oil, (2) Test- sunflower oil dosed with vitamin K2 each day. The animal treatments lasted for 17 months, and then the rats were subjected to several behavioral tests. An additional control group (3) of 10 adult rats, not aged, was also subjected to these tests. Finally, the animals were processed and the brain tissues examined.

Behavior Tests

The behavioral tests included an anxiety test, a depressive-like behavior test, and a memory test. These tests did not show any significant differences between the untreated group (3) and the MK-4 treated group (2). However, there were negative differences measured for the control- sunflower oil group (1). This group consistently had the lowest performance.

Tissue Tests

Finally, the brain tissues were collected and examined by PCR, biochemical analysis of oxidative stress, ELISA, and several histopathological and immunohistochemical processes. These tests all showed groups (1) and (3) to have similar results, while group (2) had less desirable results. This evidence all points to vitamin K2 being correlated with good brain health, preventing the signs of aging in the brain.

Vitamin K2 impact on the brain may hold promise for preserving cognitive function and decreasing age-related disorders.

Elkattawy H, Ghoneim F, Eladl M, et al. Vitamin K2 (menaquinone-7) reverses age-related structural and cognitive deterioration in naturally aging rats. Antioxidants (Basel). 2022 Mar 8;11(3):514. doi: 10.3390/antiox11030514. PMID: 35326164

Merkel cell carcinoma is a rare and aggressive skin cancer. As it begins, a painless red or blue lump in the skin forms, and then metastasizes quickly. After metastasis, patients have a terrible prognosis, with only an 18% survival rate after five years. Moreover, the incidence of this malignant carcinoma is increasing.

Currently, few therapies are available for metastatic Merkel cell carcinoma. Fortunately, immunotherapies have recently been approved as treatment, but unfortunately, most patients eventually develop resistance.

Enzyme Inhibition Tactic

Because this cancer is so difficult to treat, a team of cancer researchers in California became interested in targeting Aurora kinase A to attempt to treat this cancer. Aurora kinase A is an enzyme needed for cell proliferation, and is overexpressed in many types of cancer. In theory, by inhibiting this enzyme, the growth of the cancer will be repressed. For this reason, an inhibitor specific to Aurora kinase A , LY3295668, has recently been developed. Therefore, the researchers decided to test the effectiveness of LY3295668 against Merkel cell carcinoma in cell culture.

Cell and Animal Testing

The first test involved the application of LY3295668 to Merkel cell carcinoma cell lines. The result was that Ly3295668 inhibited the cell growth and raised levels of biomarkers of cell cycle arrest and apoptosis. Then to investigate further, mice bearing Merkel cell carcinoma tumors were created. In these mice, after treatment with LY3295668, the tumors shrank. However, when the researchers stopped LY3295668 treatment, the tumors grew again and were resistant to further LY3295668 treatment.

Insufficient Results

These results suggest that LY3295668 has the potential to treat this type of carcinoma, but that this cancer can develop resistance to this drug quickly. As noted, this chemical does show effectiveness against the cancer, however, the overall result is rather insufficient. In consequence, if LY3295668 is to be developed into a clinical treatment, it will in all likelihood need to be used in combination with other drugs.

Das B, Kannan A, Nguyen Q, et al. Selective inhibition of aurora kinase A by AK-01/LY3295668 attenuates MCC tumor growth by inducing MCC cell cycle arrest and apoptosis. Cancers (Basel). 2021 Aug;13(15):3708. doi:10.3390/cancers13153708. PMID: 34359608.

Joint replacement surgery is a common approach to treating severe joint pain or dysfunction that does not respond to less invasive therapy. Successful surgery offers improved quality of life with decreased pain and improved mobility. However, this treatment also comes with the risk of joint infection, and treatment of such an infection may not be straightforward.

New antibiotics and new combinations of antibiotics are necessary to deal with the antibiotic resistance that bacteria develop over time.

Lab Imitation of Joint Replacement

A study to mimic a joint replacement followed by infection has been carried out. In the animal lab, Wistar rats were implanted with a rod into the thigh bone. The rod was made of sterile steel and was infected with a bacterial suspension before being implanted. Two different kinds of bacteria suspension were tested.

Antibiotics Treatments Attempted

Treatment with antibiotics started seven days after the surgery. The antibiotics that were tested individually include linezolid, vancomycin, rifampin, and cotrimoxazole. A few antibiotic combinations were also tested: 1. rifampin + linezolid, 2. rifampin + vancomycin, and 3. rifampin + cotrimoxazole. After 23 days, the implants were removed, and the bacterial counts of the implant and surrounding tissues examined.

Joint Infection Under Control

In this study, rifampin is the only antibiotic to significantly reduce the bacterial infection on its own. However, it is already known that monotherapy using rifampin is not a good option. Previous studies found frequent development of resistance to treatment when only rifampin is used. An effective treatment needs to also prevent these resistance mechanisms from forming or evolving.

As it turns out, the combination antibiotic treatments used in this study were also found to be effective. And in addition, the combination treatment significantly reduced the development of resistance.

A Real Concern

Infection after joint replacement surgery is rare for a first-time surgery. However, if repeated surgeries are required, the risk of infection is significant. And since replaced joints typically last only 10-25 years, many people eventually require a second and third or more replacement surgeries. Therefore, it is important to determine an effective method of treatment for this type of infection.

Goetz J, Keyssner V, Hanses F, et al. Animal experimental investigation on the efficacy of antibiotic therapy with linezolid, vancomycin, cotrimoxazole, and rifampin in treatment of periprosthetic knee joint infections by MSRA. Bone Joint Res. 2022 Mar;11(3):143-151. doi:10.1302/2046-3758. PMID: 35227086

Background

Sotorasib and adagrasib are two new compounds that can help us to understand the mechanisms of KRAS mutations. Mutated forms of the gene KRAS encourage cell proliferation and are involved in a large fraction of all cancers. Although this gene was discovered in the 1980s, scientists have long struggled to find chemotherapies that target it. That’s because of a lack of binding sites. The new drugs sotorasib and adagrasib can bind to and inhibit one kind of mutated KRAS. Sotorasib has been approved for the treatment of non-small-cell lung cancer and adagrasib is in clinical trials.

Non-small-cell lung cancer has a history of developing resistance to drug treatments. So, it’s likely that patients may suffer resistance when sotorasib and adagrasib enter clinical use. Recently, a team of cancer researchers sought to understand resistance to these new drugs, to improve strategies to prevent it.

Experimental

The researchers exposed a cell culture line of non-small-cell lung cancer to sotorasib or adagrasib, with added N-ethyl-N-nitrosurea to encourage mutations. Then they analyzed the KRAS mutations that resulted. Five of the mutations were resistant to one drug but not the other. Therefore, sequential treatment with these drugs might help patients with this kind of resistance. Two of the KRAS mutations that emerged were resistant to both drugs. A cocktail of BI-3406 and trametinib inhibits cells with this mutation, so this cocktail might help patients who have this kind of resistance.

The mutations found in this study are not exhaustive of all the KRAS mutations. Additional mutations may appear when patients are treated with these chemicals. However, this study made good progress toward understanding the mechanisms behind KRAS chemotherapy resistance.

Koga T, Suda K, Fujino T, et al. KRAS secondary mutations that confer acquired resistance to KRAS G12C inhibitors, sotorasib and adagrasib, and overcoming strategies: insights from in vitro experiments. Journal of Thoracic Oncology. 2021 Aug;16(8):1321-1332. PMID: 33971321. doi: 10.1016/j.jtho.2021.04.015

Ovarian cancer often grows undetected into the advanced disease stages and because of this the mortality rate is relatively high. Even after using the available treatments, relapse is quite common. Recent studies of several cancers have shown positive impacts when treated with HDAC inhibitors, and even more powerful impacts when treated with an HDAC inhibitor paired with other types of inhibitors.

Cell Study

A recent study on ovarian cancer cells paired HDAC inhibitor panobinostat (LBH-589) with autophagy inhibitor chloroquine or bafilomycin A1. Together they cause DNA strand breaks and inhibit the cell’s ability to repair those breaks. The combination treatment was more powerful than the individual treatments. Discovering synergistic drug combinations such as these is the cornerstone of cancer therapy.

Animal Study

In another study, panobinostat was paired with PARP inhibitor olaparib. Mice with epithelial ovarian cancer cells were divided into 4 groups and each group subjected to one set of treatment: vehicle, panobinostat, olaparib, or panobinostat + olaparib. It was discovered that PARP inhibitors and HDAC inhibitors synergize well against HR-proficient ovarian cancer. This is exciting because HR-proficient type has proven less responsive to the developed treatments than HR-deficient type, which is more common. The overall tumor burden in the mice treated with panobinostat + olaparib was reduced. Also, there was reduced tumor proliferation and increased DNA damage observed in the harvested tumors.

Using the dual mechanisms of an HDAC inhibitor combined with another type of inhibitor is found to have a synergistic effect in many instances. The rational combination of drugs to target both cytotoxic effects and immune-modulating effects at the same time increases the potential to overcome drug resistance. This is especially important in fighting cancers such as ovarian cancer that have high mortality rates.

Ovejero-Sanchez M, Gonzalez-Sarmiento R, Herrero AB. Synergistic effect of chloroquine and panobinostat in ovarian cancer through induction of DNA damage and inhibition of DNA repair. Neoplasia. 2021 May;23(5):515-528. doi: 10.1016/j.neo.2021.04.003. PMID: 33930758

Wilson A, Gupta V, Liu Q, et al. Panobinostat enhances olaparib efficacy by modifying expression of homologous recombination repair and immune transcripts in ovarian cancer. Neoplasia. 2022 Feb;24(2):63-75. doi: 10.1016/j.neo.2021.12.002. PMID: 34933276

Vitamin K is a class of fat-soluble molecules that are required for blood clotting and calcium binding in the human body. Although acute vitamin K deficiency is rare, there is evidence that subclinical deficiency raises the risk of heart disease and osteoporosis. The menaquinones (vitamin K2) are an especially good dietary source of vitamin K because of their bioavailability and stability. Menaquinones can be found in fermented foods such as natto and dairy.

A team of researchers at the Norwegian University of Life Sciences wanted to enhance the menaquinone content of dairy to increase its functional value. To achieve this goal, they planned to genetically engineer a lactobacterium, Lactococcus lactis, that is involved in making yogurt. L. lactis contains a biosynthetic pathway to produce several menaquinones, especially menaquinone-9. The researchers tried overexpressing the genes in that pathway. Overexpression of some genes (menF, menA) caused L. lactis to produce extra menaquinone-3, instead of the more desirable menaquinone-9. The overexpression of other genes (mvk, llmg_0196) led to increased production of menaquinone-9.

When the engineered L. lactis was used to culture milk, the yogurt had enhanced levels of menaquinone-9. The researchers calculate that moderate intake of such yogurt would help many people get optimal vitamin K in their diet. This encouraging result suggests that engineered bacteria can be used more broadly to enhance nutrition.

Boe C, Holo H. Engineering Lactococcus lactis for increased vitamin K2 production. Front Bioeng Biotechnol. 2020 Mar 18;8:191. PMID: 32258010. doi: 10.3389/fbioe.2020.00191