On screening various oriental hervals which have long been used as medicines to treat some cancer-like diseases we have found in our laboratory that water extracts of the bark of $\underline{Cinnamomum}$ $\underline{cassia}$ have strong inhibitory effect on mouse leukemia L1210 cells. Later, the active substance contained in the extracts was found to be cinnamic aldehyde (CA) which is the main component of cinnamon oil. The present investigation was planned to clarify the mechanisms involved in the inhibition of CA on the growth of the L1210 cells.
The mouse leukemic cells were grown in the Fischer's medium fortified with horse serum under the presence or absence of CA for 48 hours and the effect of CA on the cell growth was observed. Similarly, cinnamic acid and cinnamic alcohol, both of which have molecular structures similar to that of CA have also been tested to find the active site of CA molecule.
Utilization of glucose as well as formation of lactate during the growth of L1210 cells were checked to see the effect of CA on the glycolysis of the cells. For the inhibitory effect on macromolecular synthesis the isotope technique was employed. That is, the use of [$^3H$]-thymidine for incorporation into DNA,[$^3H$]-uridine for incorporation into RNA, and [$^3H$]-leucine for incorporation into protein molecules.
To confirm the reaction between CHO-group of CA and SH-group of cell components the total SH-groups in culture solution treated with CA were determined. Additions of cysteine and glutathione as sources of SH-groups into CA containing buffer solution were attempted. Treatment of CA with cysteine before addition to culture broth was also tried to prevent CA-inhibition which is another proof of direct reaction between the two active groups, CHO of CA and SH of cysteine.
A continuous culture of L1210 cells in a chemostat having working volume of 420 ml was also tried in order to establish a unique culture method suitable for studying adaptation of cancer cells to chemotherapeutic agents occurring during treatments for long period.
Brief animal tests were performed using laboratory mice implanted with Sarcoma 180 in hope of possible utilization of CA in cancer chemotherapy. Comparison with an existing chemotherapeutic agent, benzaldehyde, on the inhibition of L1210 was also made.
Through the experiments performed it was found that the presence of CA in culture solution at the level of only 4.8 μg/ml could inhibit the growth of L1210 cells by 50%. The CHO-group of CA molecule was the site which inhibited the growth.
The inhibitory effect of CA on glycolysis of L1210 cells was not significant but was prominent on macromolecular synthesis with strongest inhibition on protein synthesis. The direct reaction between CHO-group of CA and SH-group of cysteine was proved. The insignificant inhibition of CA on glycolysis in which several SH-containing enzymes are involved suggested no reaction of CA with protein molecules.
From these evidences it may be concluded that CA inhibited the growth of L1210 cells by trapping SH-containing amino acid cysteine leading an imbalance in intracellular amino acid pool and in turn blocking the process of protein synthesis. The inhibition of CA on syntheses of DNA and RNA also can be explained by the disturbance of protein synthesis by the compound, i.e. blocking the synthesis of enzymes which are essential for the formation of nucleic acids.
The positive results of the animal tests together with the stronger inhibitory effects of CA than the existing chemotherapeutic agent, benzaldehyde, suggested some hope of using CA in the cancer chemotherapy.
새로운 항암제를 찾기위한 목적으로 동양에서 오랫동안 암이나 암과 유사한 질병의 치료에 이용되어 왔던 천연물을 선별 하였다. 계피 (Cinnamomum cassia) 의 물 추출물 에서 L1210 쥐 백혈구 암세포에 대하여 강력한 생장 억제 작용이 나타났으며, 계피를 여러 용매로 분획하여 조사한 결과 독성작용을 나타내는 성분은 지용성 이며, 최종적 으로 cinnamic aldehyde 임을 알게 되었다. 4.8 μg/ml 의 cinnamic aldehyde 존재하 에서 세포의 성장은 50\% 억제 되었다. Cinnamic aldehyde 는 계피의 정유 성분중 80 - 90% 를 차지하는 물질로서 현재까지 비교적 안전한 식품첨가제(방향제)로 알려져 있으며, 이러한 물질이 L1210 암세포에 대하여 강력한 세포 독성을 갖고 있다는 것은 매우 흥미있는 사실로써 저자는 cinnamic aldehyde 의 L1210 암세포에 대한 세포 독성작용 기작을 규명하고 또한 동물실험을 통하여 이 물질이 실제로 암치료에 이용될수 있는지의 가능성을 조사하였다.
Cinnamic aldehyde 는 성장에 필요한 배지의 어떤 특정 선분을 불 활성화 시키는 것이 아니라 세포 내에 들어가서 세포 내의 물질과 반응을 함으로써 세포 독성작용을 나타내는 것으로 밝혀졌으며, 따라서 cinnamic aldehyde 의 암세포 대사에 대한 영향을 조사하였다.
고분자 즉, 핵산과 단백질의 합성에 미치는 영향을 조사 하였는데 cinnamic aldehyde 는 단백질 합성과정을 가장 강력하게 억제 하였으며 31.5 μg/ml 에서 단백질 합성이 거의 완전히 중지 되었다. Cinnamic aldehyde 가 단백질 합성을 억제하는 기작을 조사하였는데 cinnamic aldehyde 의 작용 부위는 -CHO 기 인것을 알게 되었으며, 이 -CHO 기와 -SH 기를 가진 물질과의 반응을 조사 연구하였다. Cinnamic aldehyde 는 cysteine 과 매우 잘 반응을 일으키며 세포 내에서 위의 반응을 통하여 cysteine 을 고갈 시킴으로 단백질 합성을 억제 하는 데 이 억제 작용은 가역적 이었다. Cinnamic aldehyde 와 glutathione 의 반응은 거의 일어나지 않았는데 이것은 glutathione 이 구조적으로 복잡하기 때문이라고 추측되었다.
결론적으로 cinnamic aldehyde 가 세포내에 들어가서 아미노산 pool 에 있는 cysteine 과 결합하여 cysteine 을 고갈 시키며, 이로인 한 단백질 합성의 억제가 2차적으로 해당 작용과 핵산 합성을 억제하며, 따라서 생장이 억제되는 것으로 생각되었다. 동물실험을 통하여 cinnamic aldehyde 가 암 치료에 이용될 수 있다는 가능성을 얻게 되었으며, 현재 암 치료에 이용되고 있는 benzaldehyde 보다 더 강력한 작용이 있음을 알게 되었다. 또한 암세포가 cinnamic aldehyde 의 장기적인 투여에 의한 저항성 여부를 알기 위한 목적으로 연속 배양실험을 확립 하였다.
